Prognostic factor in sarcoma, and metastasis inhibitor

ABSTRACT

The present invention relates to uses of sarcopodin as a gene associated with distant metastasis or prognosis for survival in tumors. Specifically, the present invention provides a method of determining the risk of distant metastasis or prognosis for survival in a tumor, comprising measuring the expression level of sarcopodin in a tumor tissue. Furthermore, the present invention provides an agent for inhibiting metastasis containing a polynucleotide complementary to an mRNA that encodes sarcopodin or an expression vector capable of expressing the polynucleotide.

TECHNICAL FIELD

The present invention relates to uses of a gene associated with distantmetastasis of a tumor. More specifically, the present invention relatesto a method of determining the risk of distant metastasis and theprognosis for survival in spindle cell sarcoma or in colorectal cancerby utilizing the gene, a medicine for inhibiting the metastasis of asarcoma or the motility or invasive potential of sarcoma cells, a methodof screening for the medicine, and the like.

BACKGROUND ART

Malignant soft tissue tumors, particularly a class of sarcomasgenerically referred to as spindle cell sarcoma, are pathologicallyhighly diverse, and their classification remains unestablisheddefinitely. Therefore, it is considerably difficult to perform aprognostic factorial analysis by pathologic diagnostic category.Regarding the determination of the malignancy, some grading systems withcell division profiles, necrotic profiles and the like as indexes havebeen proposed, but there is no index that enables foreseeing the onsetof distant metastasis, which determines the prognosis for survival ofthe patient. For this reason, treatment by the same protocol must begiven to all cases; otherwise unnecessary adjuvant therapy is performedeven for patients with a good prognosis. Additionally, for patients witha poor prognosis, who should receive more proactive treatment, thetreatment can sometimes be inadequate. Hence, there is a demand for thedevelopment of a method of determining the prognosis for survival or therisk of onset of distant metastasis in spindle cell sarcoma.

Regarding the local recurrence of spindle cell sarcoma, prevention hasbeen increasingly possible thanks to the establishment and spread of theconcept of extensive resection. However, for the onset of distantmetastasis, which determines the prognosis for survival in spindle cellsarcoma, chemotherapy is mainly applied since the significance oftopical therapy is low. Currently, a therapeutic method using aplurality of anticancer agents in combination is used as a standardprotocol, but the response rate thereof remains up to 30%. Forimprovement of the prognosis, a therapeutic method aiming at suppressionof metastasis is thought to be theoretically the most promising; todate, however, no factor related to the metastasis of spindle cellsarcoma has been identified, so that no therapy targeting such ametastasis-related factor has been developed.

In colorectal cancer, it is routine practice that an initial diagnosisis made by fecal occult blood reactions, endoscopic examination and thelike, and a final diagnosis made by biopsy. Since a pathologic diagnosisbased on biopsy sample histology requires high skills, there is a demandfor the development of a diagnostic marker for facilitating thediagnosis thereof. Furthermore, there is another demand for thedevelopment of a method of determining the prognosis for survival or therisk of onset of distant metastasis in colorectal cancer patients.

Meanwhile, as genes that have been obtained as a result of acomprehensive cDNA acquirement examination, a gene under accessionnumber AK074185 (as a protein, FLJ00258) and a gene under accessionnumber AK094067 are registered with GenBank (Non-patent Document 1), butno report is available on the biological functions or clinical medicalsignificance thereof.

It has been reported that a protein called AFAP-110 is a substrate forpp60src bound to the actin filament, and a molecule that regulates theintegrity of the actin filament (Non-patent Documents 2 and 3). However,the involvement of AFAP-110 in cancer metastasis has not been reported.

Non-patent Document 1: Nature Genetics, vol. 36(1), p. 40-45, 2004

Non-patent Document 2: Mol. Cell Biol., vol. 13(12), p. 7892-7900, 1993

Non-patent Document 3: Oncogene, vol. 16(17), p. 2185-2195, 1998DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is a first object of the present invention to identify genesassociated with distant metastasis of tumors such as spindle cellsarcoma or the prognosis for survival in said tumors.

It is a second object of the present invention to provide a method ofdetermining the risk of onset of distant metastasis of tumors such asspindle cell sarcoma or the prognosis for survival in said tumors.

It is a third object of the present invention to provide a medicinecapable of suppressing metastases that have a major impact on theprognosis for survival in tumors such as spindle cell sarcoma.

Means for Solving the Problems

The present inventors performed a comprehensive gene expression analysison spindle cell sarcoma tissue, and attempted to identify genesassociated with the onset of distant metastasis and the occurrence oftumor-related deaths. As a result, the present inventors found that intumors wherein a functionally unknown gene registered with GenBank asAK074185 (as a protein, FLJ00258) is highly expressed, the incidence ofdistant metastasis is high and the prognosis for survival is poor.Functional analysis revealed that the gene product is a protein thatbinds to actin and Src and mediates the motility and invasive potentialof cells, and that by suppressing the expression thereof using an siRNA,the motility and invasive potential of sarcoma cells are suppressed.Furthermore, the inventors obtained a result showing that the shorterform, which consists of a carboxyl-terminal portion only, registered asAK094067, exists in this gene, which, by acting competitively on thelonger form, possibly suppresses the motility and invasive potential ofsarcoma cells.

Furthermore, analyzing the expression of this gene in colorectal mucosaltissue, it was found that in normal tissue and polyps, this gene waslittle expressed, but was expressed specifically in colorectal cancercells. In colorectal cancer cells of low heterogeneity, this geneproduct was localized in the nucleus, whereas in tumor cells of highheterogeneity, this gene product was localized in the cytoplasm; it wasfound that as the malignancy of colorectal cancer cells changed, theintracellular localization of this gene product changed.

On the basis of these findings, the present invention has beendeveloped.

Accordingly, the present invention relates to the following:

[1] A method of determining the risk of distant metastasis of a tumor,comprising measuring the expression level of any polypeptide orpolynucleotide selected from among (1) to (5) below in tumor tissue, anddetermining the risk of distant metastasis of the tumor on the basis ofthe correlation between the expression level and the incidence ofdistant metastasis:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1; and(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above.[2] The method described in [1], wherein the expression level ismeasured using any substance selected from among (i) to (iii) below:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[3] The method described in [2], wherein the substance used for themeasurement is any one selected from among (ia) to (iiia) below:(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.[4] The method described in any one of [1] to [3], wherein the tumor isspindle cell sarcoma or colorectal cancer.[5] A method of determining the prognosis for survival of a tumorpatient, comprising measuring the expression level of any polypeptide orpolynucleotide selected from among (1) to (5) below in tumor tissue, anddetermining the prognosis for survival of the tumor patient on the basisof the correlation between the expression level and the determination ofthe prognosis for survival of the tumor patient:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1; and(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above.[6] The method described in [5], wherein the expression level ismeasured using any substance selected from among (i) to (iii) below:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[7] The method described in [6], wherein the substance used for themeasurement is any one selected from among (ia) to (iiia) below:(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.[8] The method described in any one of [5] to [7], wherein the tumor isspindle cell sarcoma or colorectal cancer.[9] An agent for determining the risk of distant metastasis of a tumor,comprising any substance selected from among (i) to (iii) below:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[10] The agent described in [9], wherein the substance is any oneselected from among (ia) to (iiia) below:(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.[11] The agent described in [9] or [10], wherein the tumor is spindlecell sarcoma or colorectal cancer.[12] An agent for determining the prognosis for survival of a tumorpatient, comprising any substance selected from among (i) to (iii)below:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[13] The agent described in [12], wherein the substance is any oneselected from among (ia) to (iiia) below:(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.[14] The agent described in [12] or [13], wherein the tumor is spindlecell sarcoma or colorectal cancer.[15] A method of detecting colorectal cancer cells in colorectal mucosaltissue, comprising evaluating the expression of any polypeptide orpolynucleotide selected from among (1) to (5) below in colorectalmucosal tissue, and determining whether or not colorectal cancer cellsare contained in the tissue on the basis of the presence or absence ofthe expression:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1; and(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above.[16] An agent for detecting colorectal cancer cells in colorectalmucosal tissue, comprising any substance selected from among (i) to(iii) below:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[17] A method of determining the malignancy of a tumor cell, comprisingevaluating the localization of any polypeptide selected from among (1)to (3) below in the tumor cell, and determining the malignancy of thetumor cell on the basis of the presence or absence of the expression ofthe polypeptide in the cytoplasm:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell.[18] The method described in [17], wherein the tumor is spindle cellsarcoma or colorectal cancer.[19] An agent for determining the malignancy of a tumor cell, comprisingan antibody that specifically recognizes a polypeptide consisting of theamino acid sequence depicted in SEQ ID NO:2.[20] The agent described in [19], wherein the tumor is spindle cellsarcoma or colorectal cancer.[21] An agent for inhibiting sarcoma metastasis comprising anypolynucleotide selected from among (a) to (d) below or an expressionvector capable of expressing the polynucleotide:(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.[22] An agent for inhibiting the motility or invasive potential of asarcoma cell, comprising any polynucleotide selected from among (a) to(d) below or an expression vector capable of expressing thepolynucleotide:(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.[23] A method of screening for a substance capable of inhibiting themetastasis of a sarcoma or the motility or invasive potential of asarcoma cell, comprising the following steps of:(I) bringing into contact with each other a test substance and a cellthat permits a measurement of the expression of any polypeptide orpolynucleotide selected from among (1) to (5) below:

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell,

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell,

(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1, and

(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above;

(II) measuring the amount of any polypeptide or polynucleotide selectedfrom among (1) to (5) above expressed in the cell contacted with thetest substance, and comparing this amount expressed with the amount ofthe above-described polypeptide or polynucleotide expressed in a controlcell not contacted with the test substance; and(III) selecting a test substance that suppresses the amount of anypolypeptide or polynucleotide selected from among (1) to (5) aboveexpressed, as a substance capable of inhibiting the metastasis of asarcoma or the motility or invasive potential of a sarcoma cell, on thebasis of the results of the comparison in (II) above.[24] An agent for inhibiting the metastasis of a sarcoma, comprising anypolypeptide selected from among (1) to (3) below or an expression vectorcapable of expressing the polypeptide:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell.[25] An agent for inhibiting the motility of a sarcoma cell, comprisingany polypeptide selected from among (1) to (3) below or an expressionvector capable of expressing the polypeptide:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell.[26] A method of screening for a peptide capable of inhibiting themetastasis of a sarcoma or the motility of a sarcoma cell, comprisingthe following steps of:(I) expressing a test polypeptide comprising at least three consecutiveamino acids contained in the amino acid sequence depicted in SEQ ID NO:2in a sarcoma cell expressing a polypeptide comprising the amino acidsequence depicted in SEQ ID NO:2;(II) measuring the motility of the cell in which the test polypeptidehas been expressed, and comparing this motility with the motility of acontrol sarcoma cell in which the test polypeptide has not beenexpressed;(III) selecting a polypeptide that has suppressed the motility of thesarcoma cell as a polypeptide capable of inhibiting the metastasis of asarcoma or the motility of a sarcoma cell, on the basis of the resultsof the comparison in (II) above.[27] Any substance selected from among (i) to (iii) below fordetermining the risk of distant metastasis of a tumor:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[28] The substance described in [27], which is any one selected fromamong (ia) to (iiia) below:(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.[29] The substance described in [27] or [28], wherein the tumor isspindle cell sarcoma or colorectal cancer.[30] Any substance selected from among (i) to (iii) below fordetermining the prognosis for survival of a tumor patient:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by SEQ ID NO:2.[31] The substance described in [30], wherein the substance is any oneselected from among (ia) to (iiia) below:(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.[32] The substance described in [30] or [31], wherein the tumor isspindle cell sarcoma or colorectal cancer.[33] Any substance selected from among (i) to (iii) below, for detectingcolorectal cancer cells in colorectal mucosal tissue:(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2;(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.[34] An antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, for determining themalignancy of a tumor cell.[35] The antibody described in [34], wherein the tumor is spindle cellsarcoma or colorectal cancer.[36] Any polynucleotide selected from among (a) to (d) below or anexpression vector capable of expressing the polynucleotide, forinhibiting the metastasis of a sarcoma:(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.[37] Any polynucleotide selected from among (a) to (d) below or anexpression vector capable of expressing the polynucleotide, forinhibiting the motility or invasive potential of a sarcoma cell:(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.[38] Any polypeptide selected from among (1) to (3) below or anexpression vector capable of expressing the polypeptide, for inhibitingthe metastasis of a sarcoma:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell.[39] Any polypeptide selected from among (1) to (3) below or anexpression vector capable of expressing the polypeptide, for inhibitingthe motility of a sarcoma cell:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell.[40] A method for inhibiting the metastasis of a sarcoma in a mammal,comprising administering to the mammal an effective amount of anypolynucleotide selected from among (a) to (d) below or an expressionvector capable of expressing the polynucleotide:(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.[41] A method for inhibiting the motility or invasive potential of asarcoma cell in a mammal, comprising administering to the mammal aneffective amount of any polynucleotide selected from among (a) to (d)below or an expression vector capable of expressing the polynucleotide:(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.[42] A method for inhibiting the metastasis of a sarcoma in a mammal,comprising administering to the mammal an effective amount of anypolypeptide selected from among (1) to (3) below or an expression vectorcapable of expressing the polypeptide:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell.[43] A method for inhibiting the motility of a sarcoma cell in a mammal,comprising administering to the mammal an effective amount of anypolypeptide selected from among (1) to (3) below or an expression vectorcapable of expressing the polypeptide:(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell.

ADVANTAGES

Using the method of the present invention, it is possible to quicklydetermine the risk of onset of distant metastasis or the prognosis forsurvival in a tumor such as spindle cell sarcoma or colorectal cancerusing a convenient technique such as immunological staining using anantibody or quantitative RT-PCR using a specific probe. Using the methodof the present invention, it is possible to administer a selectivetreatment to the patient on the basis of the level of risk of onset ofdistant metastasis or the determination of prognosis for survival, thusimproving the quality of tumor treatment. Specifically, for patientsjudged to have a poor prognosis using the method of the presentinvention, an improvement of the prognosis can be aimed at byproactively performing an adjuvant therapy, and attempting for earlydetection of metastatic foci by regular diagnostic imaging. Conversely,for patients judged to have a good prognosis using the method of thepresent invention, unnecessary adjuvant therapy can be avoided. Inparticular, because the method of the present invention is applicable toall types of spindle cell sarcoma, the prognosis for survival can beaccurately determined even if a pathologic diagnosis is unestablished.

Using the method of the present invention, the presence or absence ofcolorectal cancer cells and the malignancy of colorectal cancer cellscan be determined with high accuracy.

Using the agent for inhibiting metastasis of the present invention, itis possible to selectively suppress the metastasis of spindle cellsarcoma and improve the prognosis for survival in the spindle cellsarcoma. The agent for inhibiting metastasis of the present invention isdistinctive in that the onset of distant metastasis, which is a factorthat determines the prognosis of spindle cell sarcoma, is targeted.Because the expression levels in normal tissue are much lower than thosein tumor tissue, the adverse reactions caused by expression suppressingtreatment with an siRNA are expected to be very mild, and the treatmentenables an effective treatment of spindle cell sarcoma when combinedwith currently available therapeutic methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes a cDNA microarray analysis. RNAs extracted fromspindle cell sarcoma and MSC were amplified using T7, and thereafterlabeled at the time of reverse transcription. Competitive hybridizationwas performed on 23040 kinds of genes, and the expression level of eachgene was converted to a numerical value.

FIG. 2 shows a search for genes associated with the malignancy. 65patients were divided into two groups according to the presence orabsence of each of the three events of recurrence, distant metastasis,and tumor-related death; C7059 was identified as a gene whose expressionlevel differed between the two groups.

FIG. 3 shows the expression of the C7059 gene in a cDNA microarray:patients were divided into two groups according to onset of distantmetastasis; the C7059 expression levels in the individual patients areshown. It is understood that many in the group of patients with onset ofmetastasis were positive for the expression of C7059, irrespective ofpathologic diagnosis.

FIG. 4 shows a multivariate analysis. With tumor-related death as anendpoint, a multivariate analysis was performed on nine factorsincluding C7059.

FIG. 5 shows genome and protein information on C7059.

FIG. 6 shows the genome structures of C7059 and ctC7059.

FIG. 7 shows the primary structure of the C7059 protein. Individualfunctional domains are shown. Being the shorter form, ctC7059 retainsthe protein-protein binding domain at the C-terminal.

FIG. 8 shows measurements of the amounts of C7059 and ctC7059 expressedusing quantitative RT-PCR. In the sarcoma cell line U20S, which was usedas the subject, the expression of ctC7059 was about 1/10 of that ofC7059; it is understood that in spindle cell sarcoma, the expression ofC7059 is predominant.

FIG. 9 shows a verification of microarray data using quantitativeRT-PCR. A; C7059 expression level data by quantitative RT-PCR. With theamount expressed in MSC taken as 1, C7059 expression levels wereconverted to numerical values. In addition to the patients used in themicroarray (solid circles, 44 patients), additional patients (solidsquares, 10 patients) were analyzed. B; The correlation between C7059expression level and onset of distant metastasis in the patients used inthe microarray, divided into two groups with the relative expressionlevel of 10 as the cutoff value. A significant difference in incidencewas observed between the two groups. C; An analysis on the additionalpatients. It is understood that the incidence of distant metastasis washigh in the patients with a relative expression level of 10 or more.

FIG. 10 shows how a C7059-specific polyclonal antibody was generated. A;Domestic rabbits were immunized with a fusion protein of 79 amino acidsin an amino terminal region highly specific for C7059, and not containedin ctC7059, and GST, as the antigen, to generate a polyclonal antibody.B; An about 110 kD protein was recognized by Western blot. Theexpression level agreed with the C7059 mRNA level.

FIG. 11 shows immunocytological staining. After a C7059 expressionvector was introduced into a C7059-negative sarcoma cell, the cell wasstained using an antibody. It is seen that when C7059 was stained greenwith FITC, and actin stained red with Phalloidin, both were co-localizedin some portions, with aggregation particularly in a portion where themorphology of lamellipodia was exhibited.

FIG. 12 shows immunocytological staining. The KS976 patient, whoexperienced pulmonary metastasis, was strongly positive for C7059expression mainly in the cytoplasm, and also positive in the nucleus,whereas in the KS991 patient, who was negative for distant metastasis,stainability was observed in the nucleus only.

FIG. 13 shows induction of C7059 expression by introduction of anexpression vector and suppression of C7059 expression with an siRNA. A;C7059 was integrated into a plasmid vector (PCAGGS), and the vector wasintroduced into a C7059-negative sarcoma cell, whereby good expressionof C7059 was obtained. B; Four kinds of C7059-specific siRNAs wereprepared. In particular, siRNA1-3 had a remarkable suppressive effect onC7059 expression at the mRNA and protein levels.

FIG. 14 shows the relationship between the expression of C7059 and themotility or invasive potential of cells. A; With induction of C7059expression, the invasive potential was enhanced. B; With inhibition ofC7059 expression, the motility and invasive potential decreased.

FIG. 15 shows a functional analysis of immortalized MSCs stablyexpressing C7059. With C7059 expression, both the motility (A) andinvasive potential (B) was enhanced remarkably. C; With C7059expression, the immortalized MSCs acquired an anchorage-independentgrowth potential in agar medium.

FIG. 16 shows an analysis of the binding of C7059 and the Src protein byco-immunoprecipitation. By performing immunoprecipitation with anti-FLAGantibody and Western blot with anti-Src antibody, the Src protein wasdetected (2). Conversely, by performing immunoprecipitation withanti-Src antibody and Western blot with anti-C7059 antibody (3) oranti-FLAG antibody (4), the C7059 protein was detected. Hence, it wasfound that C7059 and Src were bound together in cells.

FIG. 17 shows the confirmation of the co-localization of C7059 and Srcby immunocytological staining. In immortalized MSCs expressing C7059stably, C7059 was stained green with AF488, and Src stained red withAF555. It is understood that both were co-localized in some portions,with aggregation particularly in the lamellipodia.

FIG. 18 shows a functional analysis of ctC7059. A; ctC7059 wasintegrated into a plasmid vector (pCAGGS) to induce transientexpression. B; When ctC7059 was expressed in C7059-positive sarcomacells, the cell motility decreased.

FIG. 19 shows the expression of the C7059 protein in normal mucosa andpolyps. In the polyps, C7059 expression was observed in the cellnucleus, but not observed in the cytoplasm.

FIG. 20 shows a patient 1 (Stage IV). The expression of the C7059protein in normal mucosa and colorectal cancer is shown. The expressionof the C7059 protein was observed in the cytoplasm of colorectal cancercells, but not observed in the normal mucosa.

FIG. 21 shows a patient 2 (Stage III). A patient with hepatic metastasisand cerebral metastasis. The expression of the C7059 protein in normalmucosa and colorectal cancer is shown. The expression of the C7059protein was observed in the cytoplasm of colorectal cancer cells, butnot observed in the normal mucosa.

FIG. 22 shows a patient 3 (Stage IV). The expression of the C7059protein in normal mucosa and colorectal cancer is shown. The expressionof the C7059 protein was observed in the cytoplasm of colorectal cancercells, but not observed in the normal mucosa.

FIG. 23 shows a patient 4 (Stage IV). The expression of the C7059protein in normal mucosa and colorectal cancer is shown. The expressionof the C7059 protein was predominant in the nucleus at portion of lowheterogeneity, but predominant in the cytoplasm at invasive fronts. Inthe normal mucosa, no expression of the C7059 protein was observed.

FIG. 24 shows detection of the expression of C7059 in normal mucosa (N)and rectal or colic cancer (T) by RT-PCR. BACT: β actin.

FIG. 25 shows the correlation between disease-free survival rate andC7059 expression level (0 to 2) in Stage III rectal or colic cancerpatients.

FIG. 26 shows the correlation between disease-free survival rate andC7059 expression level (0 to 2) in patients undergoing therapeuticsurgery for extirpation of the colon.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Method of Determining theRisk of Onset of Distant Metastasis of Tumor or the Prognosis forSurvival of Tumor Patient

The present invention provides a method of determining the risk ofdistant metastasis of a tumor and the prognosis for survival of a tumorpatient, comprising measuring the expression level of any polypeptide orpolynucleotide selected from among (1) to (5) below in a tumor tissue,and determining the risk of distant metastasis of the tumor or theprognosis for survival of the tumor patient on the basis of thecorrelation between the expression level and the incidence of distantmetastasis or the prognosis for survival of the tumor patient(determination method (I) of the present invention):

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1; and(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above.

Herein, the choice of tumor is not particularly limited. As the tumor, asarcoma, colorectal cancer and the like can be mentioned. The sarcoma ispreferably spindle cell sarcoma. The colorectal cancer is preferably acancer that has developed in the colon or rectum. The determinationmethod (I) of the present invention is suitably used for spindle cellsarcoma or colorectal cancer.

The subject genes whose expression levels are measured n thedetermination method (I) of the present invention are herein genericallyreferred to as “sarcopodin”. Specifically, in the determination method(I) of the present invention, the expression level of sarcopodin in atumor tissue is measured. The sarcopodin used in the present inventionis normally of human derivation. Specifically, the polypeptides (1) to(3) above are human sarcopodin polypeptides, and the polynucleotides (4)and (5) above are polynucleotides comprising a nucleotide sequence thatencodes a human sarcopodin polypeptide.

The amino acid sequence and nucleotide sequence itself of humansarcopodin have been registered with GenBank under accession numberAK074185 (as a polypeptide, FLJ00258) and the like, and the putativegenome structures thereof have been clarified; however, no report isavailable on the biological function or clinical medical significancethereof. The present inventors have elucidated the biological functionand clinical medical significance of sarcopodin, and provide the presentinvention on the basis of the achievement.

The sarcopodin polypeptides used in the present invention possess anactivity to enhance the motility or invasive potential of a cell whenexpressed in the cell. The cell is normally derived from a mammal (forexample, rat, mouse, guinea pig, rabbit, sheep, horse, swine, bovine,monkey, human; preferably human). The cell may be a tumor cell or anon-tumor cell. The cell is preferably a mesenchymal cell. Hence, aspreferable tumor cells, sarcoma cells can be mentioned. As preferablenon-tumor cells, mesenchymal stem cells can be mentioned.

The presence or absence of such activity can be determined byintroducing an expression vector capable of expressing a desiredpolypeptide into a cell not expressing sarcopodin (preferably a sarcomacell such as Saos2), and comparing the motility or invasive potential ofthe thus-obtained cell with that of a negative control cell.

Motility means the ability of cells to move actively. Invasive potentialmeans the ability of cells to destroy the extracellular matrix andinvade the tissue. The motility of cells can be measured according to,for example, the gold colloid method. The gold colloid method is amethod wherein cells are seeded onto a cover glass with colloidal goldadhering thereto, and the area of the gold-lacking portion formed on themotion track thereof is calculated, whereby the motility of the cells ismeasured. The motility and invasive potential of cells can be evaluatedby Boyden chamber assay and the like. In Boyden chamber assay, themotility is first evaluated by the number of cells that have passed anuncoated porous PET membrane, then using a PET membrane coated with abasal membrane, the number of cells that have degraded the basalmembrane, passed the pores, and migrated to below the membrane ismeasured, and the ratio to the number of cells that have migrated ismeasured, whereby the invasive potential of the cells is evaluated.

The amino acid sequence contained in the polypeptide (2) above is anamino acid sequence having an identity of 90% or more, preferably 95% ormore, more preferably 98% or more, still more preferably 99% or more, tothe amino acid sequence depicted in SEQ ID NO:2.

Here, “identity” means a ratio (%) of identical amino acid residues toall overlapping amino acid residues in the optimal alignment where twoamino acid sequences are aligned using a mathematical algorithm known inthe technical field (preferably, the algorithm can consider introductionof gaps on one or both of the sequences for the best alignment).

Amino acid sequence identity in the present description can becalculated using the homology calculation algorithm NCBI BLAST-2(National Center for Biotechnology Information Basic Local AlignmentSearch Tool) under the following conditions (matrix=BLOSUM62; gapopen=11; gap extension=1; x_dropoff=50; expectancy=10; filtering=ON).Algorithms to determine amino acid sequence identity include, forexample, but are not limited to, the algorithm described in Karlin etal., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [the algorithm isincorporated in the NBLAST and XBLAST programs (version 2.0) (Altschulet al., Nucleic Acids Res., 25:3389-3402 (1997))], the algorithmdescribed in Needleman et al., J. Mol. Biol., 48:444-453 (1970) [thealgorithm is incorporated in the GAP program in the GCG softwarepackage], the algorithm described in Myers and Miller, CABIOS, 4:11-17(1988) [the algorithm is incorporated in the ALIGN program (version2.0), which is part of the CGC sequence alignment software package], thealgorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA,85:2444-2448 (1988) [the algorithm is incorporated in the FASTA programin the GCG software package] and the like.

The amino acid sequence contained in the polypeptide (3) above is anamino acid sequence resulting from deletion, substitution, insertion oraddition of one or a plurality of amino acids in the amino acid sequencedepicted in SEQ ID NO:2, for example, (1) an amino acid sequenceresulting from deletion of one or a plurality of (preferably 1 to 30,more preferably 1 to 10, still more preferably one to several (2 to 5))amino acids in the amino acid sequence depicted in SEQ ID NO:2, (2) anamino acid sequence resulting from addition of one or a plurality of(preferably 1 to 30, more preferably 1 to 10, still more preferably oneto several (2 to 5)) amino acids in the amino acid sequence depicted inSEQ ID NO:2, (3) an amino acid sequence resulting from insertion of oneor a plurality of (preferably 1 to 30, more preferably 1 to 10, stillmore preferably one to several (2 to 5)) amino acids in the amino acidsequence depicted in SEQ ID NO:2, (4) an amino acid sequence resultingfrom substitution of one or a plurality of (preferably 1 to 30, morepreferably 1 to 10, still more preferably one to several (2 to 5)) aminoacids in the amino acid sequence depicted in SEQ ID NO:2 by other aminoacids, or (5) an amino acid sequence being a combination of themutations (1) to (4) above (in this case, the total number of mutatedamino acids is preferably 1 to 30, more preferably 1 to 10, still morepreferably one to several (2 to 5)).

In one preferred embodiment, the polypeptides (1) to (3) above arenatural human sarcopodin polypeptides. “Natural” refers to the existenceof an amino acid sequence that constitutes a polypeptide. Natural humansarcopodin amino acid sequences include, but are not limited to, theamino acid sequence depicted in SEQ ID NO:2. It is known that apolymorphism (individual differences) usually exists ingene-constituting nucleotide sequences. Polypeptides having an aminoacid sequence different from the amino acid sequence depicted in SEQ IDNO:2, resulting from such polymorphism of the sarcopodin gene, are alsoincluded in natural human sarcopodin polypeptides, as far as theypossess an activity to enhance the motility or invasive potential of acell when expressed in the cell.

The polynucleotides (4) and (5) above are DNAs or RNAs, and arepreferably RNAs (more preferably mRNAs). As the mRNAs, mature mRNAs andinitial transcription products (immature mRNAs) that produce maturemRNAs by post-transcriptional processing can be mentioned. The mRNAs arepreferably mature mRNAs. Hence, the polynucleotides (4) and (5) abovecan be mature mRNAs of sarcopodin. As the polynucleotide of (4) above, apolynucleotide comprising the nucleotide sequence depicted in SEQ IDNO:1 can be mentioned. Nucleotide sequences are herein described as DNAsequences unless otherwise specified; however, when the polynucleotideis an RNA, thymine (T) should read as uracil (U) as appropriate.

In the determination method (I) of the present invention, the expressionlevel of a sarcopodin polypeptide or a polynucleotide (for example,mRNA) that encodes the polypeptide in a tumor tissue separated from thepatient who is the subject of measurement is measured.

The expression level of a sarcopodin polypeptide can be to measuredusing an antibody that specifically recognizes sarcopodin, for example,

(i) an antibody that specifically recognizes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2, by an immunologicaltechnique. As the immunological technique, flowcytometry analysis,radioisotope immunoassay (RIA), ELISA (Methods in Enzymol. 70: 419-439(1980)), Western blotting, immunohistochemical staining and the like canbe mentioned.

An antibody that specifically recognizes sarcopodin can be produced by aknown ordinary method of production using a sarcopodin polypeptide or apartial peptide thereof having the antigenicity as an immunogen. Asmentioned herein, antibodies include, but are not limited to, naturaltype antibodies such as polyclonal antibodies and monoclonal antibodies(mAbs), chimeric antibodies, humanized antibodies, and single-strandedantibodies that can be produced using gene recombination technology, andbinding fragments thereof. Preferably, the antibody is a polyclonalantibody, a monoclonal antibody or a binding fragment thereof. A bindingfragment means a partial region of one of the above-described antibodieshaving the specific binding activity; specifically, for example,F(ab′)₂, Fab′, Fab, Fv, sFv, dsFv, sdAb and the like can be mentioned(Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996). The classof antibody is not particularly limited; antibodies of any itotypes suchas IgG, IgM, IgA, IgD and IgE are encompassed. Preferably, the class isIgG or IgM, and in view of the ease of purification and the like, theclass is more preferably IgG.

The expression level of a polynucleotide that encodes a sarcopodinpolypeptide can be measured by a method known per se, using a nucleicacid probe or primer capable of specifically detecting thepolynucleotide, for example,

(ii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1, or(iii) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2. As examples of the measuring method, RT-PCR, Northern blotting, insitu hybridization, cDNA array and the like can be mentioned.

In one preferred embodiment, the nucleic acid probe used in thedetermination method (I) of the present invention is a polynucleotidecomprising a nucleotide sequence of about 15 bases or more, preferablyabout 18 to about 500 bases, more preferably about 18 to about 200bases, still more preferably about 18 to about 50 bases, in continuity,or a complementary sequence for the same, contained in the nucleotidesequence depicted in SEQ ID NO:1 or a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2.

In another preferred embodiment, the nucleic acid probe used in thedetermination method (I) of the present invention is a polynucleotidecomprising a nucleotide sequence that hybridizes with the nucleotidesequence depicted in SEQ ID NO:1 or a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:2 under stringent conditions. The hybridization can be performed by amethod known per se or a method based thereon, for example, a methoddescribed in Molecular Cloning, 2nd edition (J. Sambrook et al., ColdSpring Harbor Lab. Press, 1989) and the like. Examples of stringentconditions include those wherein a hybridization reaction at 45° C. in6×SSC (sodium chloride/sodium citrate) is followed by one time or moreof washing at 65° C. in 0.2×SSC/0.1% SDS and the like. Those skilled inthe art are able to easily obtain desired stringency by changing thesalt concentration of the hybridization solution, hybridization reactiontemperature, probe concentration, probe length, the number ofmismatches, hybridization reaction time, the salt concentration of thewashing solution, washing temperature and the like as appropriate. Thelength of the nucleic acid probe is normally about 15 bases or more,preferably about 18 to about 500 bases, more preferably about 18 toabout 200 bases, still more preferably about 18 to about 50 bases.

The nucleic acid probe may comprise an additional sequence (a nucleotidesequence not complementary to the polynucleotide which is the target ofdetection), as far as the specific detection is not interfered with.

The nucleic acid probe may be labeled with an appropriate labelingagent, for example, a radioisotope (e.g., ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³²P, ³³Pand the like), enzyme (e.g., β-galactosidase, β-glucosidase, alkalinephosphatase, peroxidase, malate dehydrogenase and the like), fluorescentsubstance (e.g., fluorescamine, fluorescein isothiocyanate and thelike), luminescent substance (e.g., luminol, luminol derivative,luciferin, lucigenin and the like) and the like. Alternatively, in thevicinity of a fluorescent substance (e.g., FAM, VIC and the like), aquencher (quenching substance) that absorbs the fluorescence energyproduced by the fluorescent substance may further be bound. In this modeof embodiment, upon the detection reaction, the fluorescent substanceand the quencher become separated from each other and fluorescence isdetected.

The nucleic acid primer used in the determination method (I) of thepresent invention may be any one, as far as it has been designed toenable specific amplification of a portion or the entire region of apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1 or a nucleotide sequence that encodes a polypeptide consisting ofthe amino acid sequence depicted in SEQ ID NO:2. For example, a pair ofpolynucleotides can be mentioned which is a combination of apolynucleotide comprising a nucleotide sequence as long as about 15 toabout 50 bases, preferably about 18 to about 30 bases, which hybridizeswith a portion of a complementary sequence for the above-describednucleotide sequence, and a polynucleotide comprising a nucleotidesequence as long as about 15 to about 50 bases, preferably about 18 toabout 30 bases, which hybridizes with a portion of the above-describednucleotide sequence on the 3′ side from said hybridization site, whereinthe lengths of nucleic acid fragments amplified thereby are about 50 toabout 1,000 bases, preferably about 50 to about 500 bases, morepreferably about 50 to about 200 bases.

The nucleic acid primer may comprise an additional sequence (anucleotide sequence not complementary to the polynucleotide which is thetarget of detection), as far as the specific detection is not interferedwith.

The nucleic acid primer may be labeled with an appropriate labelingagent, for example, a radioisotope (e.g., ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³²P, ³³Pand the like), enzyme (e.g., β-galactosidase, β-glucosidase, alkalinephosphatase, peroxidase, malate dehydrogenase and the like), fluorescentsubstance (e.g., fluorescamine, fluorescein isothiocyanate and thelike), luminescent substance (e.g., luminol, luminol derivative,luciferin, lucigenin and the like) and the like.

The nucleic acid probe or primer may be a DNA or an RNA, and may besingle-stranded or double-stranded. In the case of a double strand, thenucleic acid may be a double-stranded DNA, a double-stranded RNA, or aDNA/RNA hybrid. Therefore, when a nucleic acid having a certainnucleotide sequence is described herein, it should be understood thatthe term nucleic acid is used with a meaning that encompasses all ofsingle-stranded polynucleotides having the nucleotide sequence,single-stranded polynucleotides having a sequence complementary to thenucleotide sequence, and double-stranded polynucleotides which arehybrids thereof unless otherwise stated.

The above-described nucleic acid probe or primer can be synthesized, forexample, using an automated DNA/RNA synthesizer according to aconventional method, on the basis of information on the nucleotidesequence depicted in SEQ ID NO:1.

The sarcopodin gene has a second transcription initiation point in the9th intron thereof; this second transcription produces the shorter form,which has a structure wherein the N-terminal region of the sarcopodindescribed above (hereinafter sometimes referred to as the longer form)is lacked (FIG. 6). For example, as a shorter form human sarcopodin, apolypeptide consisting of the amino acid sequence depicted in SEQ IDNO:4 encoded by the nucleotide sequence depicted in SEQ ID NO:3 (codingregion: nucleotide numbers 317 to 1468) can be mentioned. This shorterform human sarcopodin lacks 385 amino acids at the N-terminus of thehuman sarcopodin consisting of the amino acid sequence depicted in SEQID NO:2, and consists of the remainder (the amino acid sequence shown byamino acid numbers 386 to 768 in SEQ ID NO:2). Therefore, in thedetermination method (I) of the present invention, when an antibody thatspecifically recognizes a region shared by the longer form and theshorter form of sarcopodin, or a nucleic acid probe or primer thatspecifically detects a shared region, is used, the sum of the amount ofthe longer and shorter forms expressed will be measured. Such cases alsofall within the scope of the present invention, as far as the resultingexpression level measured includes the expression level of anypolypeptide or polynucleotide selected from among (1) to (5) above.

As shown in Examples below, the expression of the longer form ispredominant over the expression of the shorter form in tumors(particularly spindle cell sarcoma). Therefore, by specificallydetecting the longer form of sarcopodin, the risk of distant metastasisor prognosis for survival in a tumor can be determined with highersensitivity. Hence, in one preferred embodiment, of the antibodies,nucleic acid probes or primers in (i) to (iii) above, one capable ofspecifically detecting a region that is present in the longer form, butnot present in the shorter form, is used in measuring the expressionlevel. As such antibodies, nucleic acid probes and primers, (ia) to(iiia) below can be mentioned:

(ia) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iia) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.

Of them, the antibodies, nucleic acid probes or primers in (ib) to(iiib) below are preferable because they recognize a region having asequence of particularly high specificity for longer-form sarcopodin.

(ib) an antibody that specifically recognizes a polypeptide consistingof the amino acid sequence depicted in SEQ ID NO:2, a recognitionepitope for the antibody being present within the region shown by aminoacid numbers 1 to 385 in SEQ ID NO:2;(iib) a nucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence shown by nucleotidenumbers 117 to 413 in SEQ ID NO:1; and(iiib) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 35 to 113 in SEQ ID NO:2.

Next, on the basis of the expression level of sarcopodin measured, therisk of distant metastasis of a tumor or the prognosis for survival of atumor patient is determined. As shown in Examples below, the higher theexpression level of sarcopodin in a tumor tissue is, the higher theincidence of distant metastasis is, and the prognosis for survival ofthe patient is worse. The above-described determination is made on thebasis of such a correlation between the expression level of sarcopodinand the incidence of distant metastasis of the tumor or the prognosisfor survival of the tumor patient.

For example, tumors are separated from a patient having a good prognosisfor survival without distant metastasis (negative control) and a patienthaving a poor prognosis for survival with distant metastasis (positivecontrol), the sarcopodin expression levels in the tumors separated fromthe subject patients are compared with those of a positive control and anegative control. Alternatively, a diagram of the correlation betweenthe sarcopodin expression level in the tumors and the incidence ofdistant metastasis or prognosis for survival (e.g., length of survival)may be prepared in advance, and the sarcopodin expression levels in thetumors separated from the subject patients may be compared with thediagram of the correlation. The comparison of the expression levels ispreferably made on the basis of the presence or absence of a significantdifference.

When the sarcopodin expression level in the subject of measurement isrelatively high, judging from the results of the comparison ofsarcopodin expression levels, it can be determined that the risk ofonset of distant metastasis of a tumor is relatively high, or theprognosis for survival of the patient is relatively poor. Conversely,when the sarcopodin expression level in the subject of measurement isrelatively low, it can be determined that the risk of onset of distantmetastasis of a tumor is relatively low, or the prognosis for survivalof the patient is relatively good.

The determination can also be made by previously setting a cutoff valuefor sarcopodin expression levels, and comparing the sarcopodinexpression level measured and this cutoff value. For example, when thesarcopodin expression level measured is not less than the aforementionedcutoff value, it can be determined that the risk of onset of distantmetastasis of a tumor is relatively high, or the prognosis for survivalof the patient is relatively poor. Conversely, when the sarcopodinexpression level is lower than the cutoff value, it can be determinedthat the risk of onset of distant metastasis of a tumor is relativelylow, or the prognosis for survival of the patient is relatively good. “Acutoff value” is a value that meets the requirements for both highdiagnostic sensitivity (true positive rate) and high diagnosticspecificity (true negative rate) when a disease or condition is judgedwith that value as a criterion. For example, a sarcopodin expressionlevel that produces high positive rates in subjects who have experienceddistant metastasis or subjects whose survival is not longer than a givenlength, and high negative rates in subjects who have not experienceddistant metastasis or subjects whose survival exceeds the given length,can be set as a cutoff value.

The present invention also provides an agent for determining the risk ofdistant metastasis of a tumor or the prognosis for survival of a tumorpatient, containing any substance selected from among (i) to (iii) above(referred to as the agent (I) of the present invention). The substanceis preferably any substance selected from among (ia) to (iiia) above,more preferably any substance selected from among (ib) to (iiib) above.The agent (I) of the present invention can be a kit for determining therisk of distant metastasis of a tumor or the prognosis for survival of atumor patient. Using the agent (I) of the present invention, the risk ofdistant metastasis of a tumor or the prognosis for survival of a tumorpatient can easily be determined by the above-described method.

The substances (i) to (iii) can be separately (or in a mixed form ifpossible) dissolved in water or an appropriate buffer solution (e.g., TEbuffer, PBS and the like) to obtain an appropriate concentration, andpreserved at about −20° C. to 4° C.

The agent (I) of the present invention may further contain otheringredients necessary for the implementation of the method of measuringsarcopodin expression levels as constituents thereof according to themethod.

For example, provided that the agent (I) of the present inventioncontains the substance (i) above, the risk of distant metastasis orprognosis for survival in spindle cell sarcoma can be determined bymeasuring the sarcopodin expression level by immunological technique. Inthis case, the agent (I) of the present invention can further contain alabeled secondary antibody, color developing substrate, blocking liquid,washing buffer solution, ELISA plate, blotting membrane and the like.

Provided that the agent (I) of the present invention contains thesubstance (ii) or (iii) above, the risk of distant metastasis of a tumoror the prognosis for survival of a tumor patient can be determined bymeasuring sarcopodin expression level by RT-PCR, Northern blotting, insitu hybridization, cDNA array and the like. When RT-PCR is used for themeasurement, the agent (I) of the present invention may further containa 10×PCR reaction buffer solution, 10×MgCl₂ aqueous solution, 10×dNTPsaqueous solution, Taq DNA polymerase (5 U/μL), reverse transcriptase andthe like. When Northern blotting or cDNA array is used for themeasurement, the agent (I) of the present invention can further containa blotting buffer solution, labeling reagent, blotting membrane and thelike. When in situ hybridization is used for the measurement, the agent(I) of the present invention can further contain a labeling reagent,color developing substrate and the like.

2. Method of Detecting Colorectal Cancer Cells

As shown in Examples below, in colorectal mucosal tissue, sarcopodin islittle expressed in normal tissue and polyps, whereas in colorectalcancer cells, sarcopodin is specifically expressed. Therefore, byevaluating the expression of sarcopodin in colorectal mucosal tissue,and analyzing for the presence or absence of the expression thereof,colorectal cancer cells can be detected in colorectal mucosal tissue.

Accordingly, the present invention provides a method of detectingcolorectal cancer cells in colorectal mucosal tissue, comprisingevaluating the expression of any polypeptide or polynucleotide selectedfrom among (1) to (5) below in colorectal mucosal tissue, anddetermining whether or not colorectal cancer cells are contained in thetissue on the basis of the presence or absence of the expression(detection method (I) of the present invention):

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1; and(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above.

The definitions for the polypeptides or polynucleotides (1) to (5) aboveare the same as those described in the (1. Method of determining therisk of onset of distant metastasis of tumor or the prognosis forsurvival of tumor patient) section.

In the detection method (I) of the present invention, the expression ofa sarcopodin polypeptide or a polynucleotide (for example, mRNA) thatencodes the polypeptide in colorectal mucosal tissue separated from apatient who is the subject of measurement is evaluated.

The expression of the sarcopodin polypeptide can be evaluated accordingto the method described in detail in the (1. Method of determining therisk of onset of distant metastasis of tumor or the prognosis forsurvival of tumor patient) section, using the antibody, nucleic acidprobe, primer and the like described in (i) to (iii) above, preferably(ia) to (iiia) above, more preferably (ib) to (iiib) above.

Next, on the basis of the presence or absence of the expression ofsarcopodin in the colorectal mucosal tissue, whether or not colorectalcancer cells are present in the tissue is determined.

When a cell expressing sarcopodin is found in the colorectal mucosaltissue from the subject of measurement, it can be determined that theprobability of the presence of colorectal cancer cells in the tissue ishigh. Conversely, when no cell expressing sarcopodin is found in thecolorectal mucosal tissue from the subject of measurement, it can bejudged that the probability of the presence of colorectal cancer cellsin the tissue is low.

The present invention also provides an agent for detecting colorectalcancer cells in colorectal mucosal tissue, containing any substanceselected from among (i) to (iii) above (referred to as the agent (II) ofthe present invention). The substance is preferably any substanceselected from among (ia) to (iiia) above, more preferably any substanceselected from among (ib) to (iiib) above. The agent (II) of the presentinvention) can be a kit for detecting colorectal cancer cells incolorectal mucosal tissue. Using the agent (II) of the presentinvention, colorectal cancer cells in colorectal mucosal tissue caneasily be detected by the above-described method.

The substances (i) to (iii) can be separately (or in a mixed form ifpossible) dissolved in water or an appropriate buffer solution (e.g., TEbuffer, PBS and the like) to obtain an appropriate concentration, andpreserved at about −20° C. to 4° C.

The agent (II) of the present invention may further contain otheringredients necessary for the implementation of a method of evaluationof sarcopodin expression as constituents thereof according to themethod.

For example, provided that the agent (II) of the present inventioncontains the substance (i) above, the presence or absence of colorectalcancer cells in colorectal mucosal tissue can be determined byevaluating the sarcopodin expression by an immunological technique. Inthis case, the agent (II) of the present invention can further contain alabeled secondary antibody, color developing substrate, blocking liquid,washing buffer solution, ELISA plate, blotting membrane and the like.

Provided that the agent (II) of the present invention contains thesubstance (ii) or (iii) above, the presence or absence of colorectalcancer cells in colorectal mucosal tissue can be determined by measuringsarcopodin expression level by RT-PCR, Northern blotting, in situhybridization, cDNA array and the like. When RT-PCR is used for themeasurement, the agent (I) of the present invention can further containa 10×PCR reaction buffer solution, 10×MgCl₂ aqueous solution, 10×dNTPsaqueous solution, Taq DNA polymerase (5 U/μL), reverse transcriptase andthe like. When Northern blotting or cDNA array is used for themeasurement, the agent (II) of the present invention can further containa blotting buffer solution, labeling reagent, blotting membrane and thelike. When in situ hybridization is used for the measurement, the agent(II) of the present invention can further contain a labeling reagent,color developing substrate and the like.

3. Method of Determining the Malignancy of Tumor Cell

As shown in Examples below, in spindle cell sarcoma cells withoutdistant metastasis and colorectal cancer cells of low heterogeneity,sarcopodin was localized in the nucleus, whereas in spindle cell sarcomacells with distant metastasis and tumor cells of high heterogeneity,sarcopodin was also localized in the cytoplasm; it is realized that asthe malignancy of tumor cells changes, the intracellular localization ofsarcopodin changes. Therefore, by evaluating the localization ofsarcopodin in a tumor cell, and analyzing for the presence or absence ofthe expression in the cytoplasm thereof, the malignancy of the tumorcell can be determined.

Accordingly, the present invention provides a method of determining themalignancy of a tumor cell, comprising evaluating the localization ofany polypeptide selected from among (1) to (3) below in the tumor cell,and determining the malignancy of the tumor cell on the basis of thepresence or absence of the expression of the polypeptide in thecytoplasm (determination method (II) of the present invention):

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell.

The definitions for the polypeptides (1) to (3) above are the same asthose described in the (1. Method of determining the risk of distantmetastasis of tumor or the prognosis for survival of tumor patient)section.

In the determination method (II) of the present invention, thelocalization of a sarcopodin polypeptide in a tumor cell contained in atumor tissue separated from a patient who is the subject of measurementis evaluated.

The intracellular localization of the sarcopodin polypeptide can beevaluated using the antibody described in (i) above, preferably (ia)above, more preferably (ib) above, by immunohistochemical staining.

Next, the malignancy of a tumor cell is determined on the basis of thepresence or absence of the expression of sarcopodin in the cytoplasm.When sarcopodin expression is found in the cytoplasm of a tumor cellfrom the subject of measurement, the tumor cell can be determined tohave a high malignancy. Conversely, when sarcopodin expression is notobserved in the cytoplasm of the tumor cell from the subject ofmeasurement, but remains in the nucleus, or when sarcopodin expressionis not observed in any site in the tumor cell from the subject ofmeasurement, the tumor cell can be determined to have a low malignancy.

The present invention also provides an agent for determining themalignancy of a tumor cell, containing the antibody described in (i)above (referred to as the agent (III) of the present invention). Theantibody is preferably the antibody described in (ia) above, morepreferably the antibody described in (ib) above. The agent (III) of thepresent invention can be a kit for determining the malignancy of a tumorcell. Using the agent (III) of the present invention, the malignancy ofa tumor cell can easily be determined by the method described above.

The antibody described in (i) above can be dissolved in water or anappropriate buffer solution (e.g., TE buffer, PBS and the like) toobtain an appropriate concentration, and preserved at about −20° C. to4° C.

The agent (III) of the present invention may further contain otheringredients necessary for the implementation of a method of evaluationof sarcopodin expression as constituents thereof according to themethod. For example, the agent (III) of the present invention canfurther contain a labeled secondary antibody, color developingsubstrate, blocking liquid, washing buffer solution and the like thatare useful in immunohistochemical staining.

4. Agent for Inhibiting the Metastasis of Sarcoma or the Motility orInvasive Potential of Sarcoma Cells

The present invention provides an agent for inhibiting the metastasis ofa sarcoma or the motility or invasive potential of a sarcoma cell,containing a substance that inhibits the expression or function ofsarcopodin (inhibitor (I) of the present invention). In a preferredembodiment of the substance, the following polynucleotides can bementioned:

(A) a polynucleotide (DNA or RNA) comprising a nucleotide sequencecomplementary to the nucleotide sequence of an mRNA (mature mRNA orinitial transcription product) that encodes sarcopodin or a partialsequence thereof as long as 15 bases or more; and(B) a polynucleotide (DNA or RNA) comprising a nucleotide sequencehybridizable with an mRNA (mature mRNA or initial transcription product)or chromosome DNA that encodes sarcopodin under physiological conditionsfor an animal (preferably human) which is the subject of treatment, andsuppressing the transcription or translation of sarcopodin in thehybridized state.

The polynucleotides (A) and (B) above are specifically identified asfollows:

(a) a polynucleotide comprising a nucleotide sequence complementary tothe nucleotide sequence depicted in SEQ ID NO:1, the nucleotide sequenceof an initial transcription product that produces the former nucleotidesequence by post-transcriptional processing, or a partial sequencethereof as long as 15 bases or more;(b) a polynucleotide comprising a nucleotide sequence complementary to anucleotide sequence that encodes any polypeptide selected from among (1)to (3) below, the nucleotide sequence of an initial transcriptionproduct that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell;

(c) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and(d) a polynucleotide comprising a nucleotide sequence hybridizable,under physiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of a nucleotide sequencethat encodes any polypeptide selected from among (1) to (3) above, or aninitial transcription product that produces the former nucleotidesequence by post-transcriptional processing, and suppressing thetranscription or translation of any one of the polypeptides (1) to (3)in the hybridized state.

The definitions for the polypeptides (1) to (3) above are the same asthose described in the (1. Method of determining the risk of distantmetastasis or prognosis for survival in spindle cell sarcoma, and agenttherefor) section. The polynucleotides (a) to (d) above are useful asantisense nucleic acids or siRNAs against sarcopodin. Embodiments of thepolynucleotides (a) to (d) above can be chosen as appropriate accordingto the intended use thereof.

“An antisense nucleic acid” refers to a nucleic acid comprising anucleotide sequence hybridizable with a target mRNA (mature mRNA orinitial transcription product) under physiological conditions for thecells that express the target mRNA, and being capable of inhibiting thetranslation of the polypeptide encoded by the target mRNA in thehybridized state. The antisense nucleic acid may be a DNA or an RNA, andmay be a DNA/RNA chimera. Since natural-form antisense nucleic acidshave the phosphodiester linkages thereof decomposed readily by nucleasesbeing present in the cells, the antisense nucleic acid of the presentinvention can also be synthesized using a modified nucleotide such asthe thiophosphate form (phosphate bond P═O is replaced with P═S) or the2′-O-methyl form, which are stable to the nucleases. Other factorsimportant for the design of the antisense nucleic acid includeincreasing the water solubility and cell membrane permeability and thelike; these can also be achieved by improving dosage forms, such as theuse of liposomes or microspheres.

The length of the portion that hybridizes with the target mRNA in theantisense nucleic acid is not particularly limited, as far as theportion is hybridizable specifically with a mature mRNA or initialtranscription product of sarcopodin, and capable of inhibiting thetranslation of the sarcopodin polypeptide in the hybridized state; thelength is about 15 bases for the shortest, and as long as thefull-length sequence of the mRNA (mature mRNA or initial transcriptionproduct) for the longest. In view of hybridization specificity, thelength of the portion that hybridizes with the target mRNA is, forexample, about 15 bases or more, preferably about 18 bases or more, morepreferably about 20 bases or more. In view of the ease of synthesis,antigenicity issues and the like, the length of the portion thathybridizes with the target mRNA is, for example, about 200 bases orless, preferably about 50 bases or less, more preferably about 30 basesor less. Hence, the length of the portion that hybridizes with thetarget mRNA is, for example, about 15 to about 200 bases, preferablyabout 18 to about 50 bases, more preferably about 20 to about 30 bases.

The target nucleotide sequence for the antisense nucleic acid is notparticularly limited, as far as it is a sequence such that thetranslation of sarcopodin is inhibited when hybridizing with theantisense nucleic acid; the sequence may be the full-length sequence ofan mRNA (mature mRNA or initial transcription product) of sarcopodin ora partial sequence thereof (for example, about 15 bases or more,preferably about 18 bases or more, more preferably about 20 bases ormore), or an intron portion of the initial transcription product;however, when an oligonucleotide is used as the antisense nucleic acid,it is desirable that the target sequence be located between the 5′-endof the mRNA of sarcopodin and the C-terminal of the coding region.

The nucleotide sequence of the portion that hybridizes with the targetmRNA in the antisense nucleic acid varies depending on the basecomposition of the target sequence, and has an identity of normallyabout 90% or more (preferably 95% or more, most preferably 100%) to thecomplementary sequence for the target sequence, so as to be capable ofhybridizing with the sarcopodin mRNA under physiological conditions.Identity in nucleotide sequence can be calculated using, for example,the homology calculation algorithm NCBI BLAST-2 (National Center forBiotechnology Information Basic Local Alignment Search Tool) under thefollowing conditions (gap open=5; gap extension=2; x_dropoff=50;expectancy=10; filtering=ON).

The size of the antisense nucleic acid is normally about 15 bases ormore, preferably about 18 bases or more, more preferably about 20 basesor more. In view of the ease of synthesis, antigenicity issues and thelike, the size is normally about 200 bases or less, preferably about 50bases or less, more preferably about 30 bases or less.

Furthermore, the antisense nucleic acid may be one not only capable ofhybridizing with the sarcopodin mRNA or initial transcription productthereof to inhibit the translation, but also capable of binding to thesarcopodin gene, which is a double-stranded DNA, to form a triplex andinhibit the transcription into mRNA.

An siRNA against sarcopodin is a double-stranded oligo-RNA comprising anucleotide sequence complementary to the nucleotide sequence ofsarcopodin mRNA (mature mRNA or initial transcription product) or apartial sequence thereof (preferably in the coding region) (in the caseof the initial transcription product, an intron portion is included).What is called RNA interference (RNAi), a phenomenon in whichtransferring a short double-stranded RNA into a cell results in thedegradation of mRNAs that are complementary to the RNA, has long beenknown to occur in nematodes, insects, plants and the like; recently,this phenomenon was confirmed as occurring also in animal cells [Nature,411(6836): 494-498 (2001)], and this is attracting attention as aribozyme-substitute technique.

A representative siRNA is a double-stranded oligo-RNA consisting of anRNA having a sequence complementary to the nucleotide sequence of themRNA of the target gene or a partial sequence thereof (hereinafter,target nucleotide sequence) and a complementary strand for the RNA. Asingle-stranded RNA wherein a sequence (first sequence) complementary tothe target nucleotide sequence and a complementary sequence (secondsequence) for the same are linked via a hairpin loop portion, andwherein the first sequence forms a double-stranded structure with thesecond sequence by assuming a hairpin loop structure (small hairpin RNA:shRNA), also represents a preferred embodiment of siRNA.

The length of the portion complementary to the target nucleotidesequence, contained in the siRNA, is normally about 15 bases or more,preferably 18 bases or more, more preferably about 20 bases(representatively about 21 to 23 bases long), but is not particularlylimited, as far as RNA interference can be caused. When the siRNA islonger than 23 bases, the siRNA undergoes degradation in cells toproduce an siRNA about 20 bases long; therefore, theoretically, theupper limit of the portion complementary to the target nucleotidesequence is the full length of the nucleotide sequence of an mRNA(mature mRNA or initial transcription product) of the target gene.However, in view of the ease of synthesis, antigenicity issues and thelike, the length of the complementary portion is, for example, about 200bases or less, preferably about 50 bases or less, more preferably about30 bases or less. Hence, the length of the complementary portion is, forexample, about 15 bases or more, preferably about 18 to about 200 bases,more preferably about 20 to about 50 bases, still more preferably about20 to about 30 bases.

The length of the siRNA is normally about 18 bases or more, for example,about 20 bases (representatively about 21 to 23 bases long), but is notparticularly limited, as far as RNA interference can be caused; there istheoretically no upper limit on the length of the siRNA. However, inview of the ease of synthesis, antigenicity issues and the like, thelength of the siRNA is, for example, about 200 bases or less, preferablyabout 50 bases or less, more preferably about 30 bases. Hence, thelength of the siRNA is, for example, about 18 bases or more, preferablyabout 18 to about 200 bases, more preferably about 20 to about 50 bases,still more preferably about 20 to about 30 bases. The length of theshRNA is expressed as the length of the double-stranded moiety when theshRNA assumes a double-stranded structure.

It is preferable that the target nucleotide sequence and the sequencecomplementary thereto contained in the siRNA be completely complementaryto each other. However, in the presence of a base mutation at a positionapart from the center of the siRNA (the mutation can be fall in therange of identity of at least 90% or more, preferably 95% or more), thecleavage activity by RNA interference is not completely lost, but apartial activity can remain. On the other hand, a base mutation in thecenter of the siRNA has a major influence to the extent that canextremely reduce the mRNA cleavage activity by RNA interference.

The siRNA may have an additional base that does not form a base pair atthe 5′- and/or 3′-end. The length of the additional base is normally 5bases or less. Although the additional base may be a DNA or an RNA, useof a DNA makes it possible to improve the stability of the siRNA.Examples of the sequences of such additional bases include, but are notlimited to, the sequences ug-3′, uu-3′, tg-3′, tt-3′, ggg-3′, guuu-3′,gttt-3′, ttttt-3′, uuuuu-3′ and the like.

The length of the loop portion of the hairpin loop of the shRNA is notparticularly limited, as far as RNA interference can be caused; thelength is normally about 5 to 25 bases. The nucleotide sequence of theloop portion is not particularly limited, as far as a loop can beformed, and the shRNA can cause RNA interference.

The polynucleotide described above can be prepared by determining thetarget sequence on the basis an mRNA sequence (for example, thenucleotide sequence depicted in SEQ ID NO:1) or chromosome DNA sequenceof sarcopodin, and synthesizing a nucleotide sequence complementarythereto using a commercially available automated DNA/RNA synthesizer(Applied Biosystems, Beckman and the like). An siRNA can be prepared byseparately synthesizing a sense strand and antisense strand using anautomated DNA/RNA synthesizer, denaturing the strands in an appropriateannealing buffer solution at about 90 to about 95° C. for about 1minute, and then performing annealing at about 30 to about 70° C. forabout 1 to about 8 hours. A longer double-stranded polynucleotide can beprepared by synthesizing complementary oligonucleotide strands in a waysuch that they overlap with each other, annealing the strands, and thenperforming ligation with a ligase.

The inhibitor (I) of the present invention may have an expression vectorcapable of expressing (encoding) any polynucleotide selected from among(a) to (d) above as an active ingredient thereof. In the expressionvector, the polynucleotide described above or a nucleic acid (preferablyDNA) that encodes the same is operably linked to a promoter capable ofexhibiting promoter activity in cells (for example, sarcoma cells) of amammal (preferably human) which is the subject of administration.

The promoter used is not particularly limited, as far as it is capableof functioning in cells of the mammal which is the subject ofadministration. As the promoter, pol I promoter, pol II promoter, polIII promoter and the like can be used. Specifically, viral promoterssuch as the SV40-derived initial promoter and cytomegalovirus LTR,mammalian constitutive protein gene promoters such as the β-actin genepromoter, and RNA promoters such as the tRNA promoter, and the like areused.

When the expression of an siRNA is intended, it is preferable that a polIII promoter be used as the promoter. As examples of the pol IIIpromoter, the U6 promoter, H1 promoter, tRNA promoter and the like canbe mentioned.

An expression vector of the present invention preferably comprises atranscription termination signal, i.e., a terminator region, downstreamof the above-described polynucleotide or nucleic acid that encodes thesame. Furthermore, a selection marker gene for selection of transformantcells (a gene that confers resistance to a drug such as tetracycline,ampicillin, or kanamycin, a gene that compensates for an auxotrophicmutation and the like) can further be contained.

Although there is no limitation on the choice of expression vector usedin the present invention, suitable vectors for administration to mammalssuch as humans include viral vectors such as retrovirus, adenovirus, andadeno-associated virus. Adenovirus, in particular, has advantages suchas very high gene transfer efficiency and transferability tonon-dividing cells as well. However, because the integration oftransgenes into host chromosomes is extremely rare, the gene expressionis transient and normally persists only for about 4 weeks. Consideringthe persistence of therapeutic effect, it is also preferable to useadeno-associated virus, which offers a relatively high efficiency ofgene transfer, which can be transferred into non-dividing cells as well,and which can be integrated into chromosomes via an inverted terminalrepeat (ITR).

Administration of the inhibitor (I) of the present invention, whichcomprises the polynucleotide described above or an expression vectorcapable of expressing the same as an active ingredient thereof, isperformed by an in vivo method wherein the transfer is achieved bydirectly administering the vector into the body of the subject ofadministration. In this case, the viral vector is administeredintravenously, intra-arterially, subcutaneously, intradermally,intramuscularly, intraperitoneally or the like in the form of aninjection. If the production of a neutralizing antibody against theviral vector is problematic, topically injecting the vector near theaffected site (sarcoma tissue) (in situ method) can lessen the adverseeffect of the presence of the antibody.

The inhibitor (I) of the present invention can comprise, in addition tothe above-described polynucleotide or an expression vector capable ofexpressing the same, an optionally chosen carrier, for example, apharmaceutically acceptable carrier.

As examples of the pharmaceutically acceptable carrier, excipients suchas sucrose and starch; binders such as cellulose and methylcellulose;disintegrants such as starch and carboxymethylcellulose; lubricants suchas magnesium stearate and Aerosil; flavoring agents such as citric acidand menthol; preservatives such as sodium benzoate and sodium hydrogensulfite; stabilizers such as citric acid and sodium citrate; suspendingagents such as methylcellulose and polyvinylpyrrolidone; dispersingagents such as surfactants; diluents such as water and physiologicalsaline; base waxes; and the like can be mentioned, which, however, arenot to be construed as limiting.

To promote the introduction of a polynucleotide into cells, theinhibitor (I) of the present invention can further comprise a reagentfor nucleic acid transfection. When the polynucleotide is integrated ina viral vector, particularly a retrovirus vector, retronectin,fibronectin, polybrene or the like can be used as the transfectionreagent. When the polynucleotide is integrated in a plasmid vector, acationic lipid such as lipofectin, lipofectamine, DOGS (transfectam),DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethylenimine) (PEI)can be used.

As preparations suitable for oral administration, liquids, capsules,sachets, tablets, suspensions, emulsions and the like can be mentioned.

Preparations suitable for parenteral administration (for example,subcutaneous injection, intramuscular injection, topical injection,intraperitoneal administration and the like) include aqueous andnon-aqueous isotonic sterile injectable liquids, which may contain anantioxidant, a buffer solution, a bacteriostatic agent, an isotonizingagent and the like. Aqueous and non-aqueous sterile suspensions can alsobe mentioned, which may contain a suspending agent, a solubilizer, athickening agent, a stabilizer, an antiseptic and the like. Thesepreparations can be encapsulated in containers such as ampoules andvials for unit dosage or a plurality of dosages. It is also possible tofreeze-dry the active ingredient and a pharmaceutically acceptablecarrier, and store the preparation in a state that may be dissolved orsuspended in an appropriate sterile vehicle just before use.

The content of the polynucleotide described above in the pharmaceuticalcomposition is, for example, about 0.1 to 100% by weight of the entirepharmaceutical composition.

Although the dosage of the inhibitor (I) of the present invention variesdepending on the activity and choice of the active ingredient,seriousness of illness, recipient animal species, the recipient's drugreceptivity, body weight, age, and the like, and cannot be generalized,the dosage is normally about 0.001 to about 500 mg/kg, based on theamount of active ingredient, per day for an adult.

The inhibitor (I) of the present invention is preferably safelyadministered to a mammal (for example, rat, mouse, guinea pig, rabbit,sheep, horse, swine, bovine, monkey, human) in a way such that theabove-described polynucleotide or expression vector capable ofexpressing the same, which is the active ingredient thereof, will bedelivered to a sarcoma tissue (sarcoma cells).

Because the above-described polynucleotide possesses an activity toinhibit the motility or invasive potential of sarcoma cells, metastasisof a sarcoma (particularly spindle cell sarcoma) can be inhibited toimprove the prognosis for survival by administering the inhibitor (I) ofthe present invention to a patient with a sarcoma (particularly spindlecell sarcoma) or a patient after treatment of a sarcoma at a risk ofdistant metastasis of the sarcoma and the like.

5. Screening Method (I) for Substance Capable of Inhibiting SarcomaMetastasis and the Like

The present invention also provides a method of screening for asubstance capable of inhibiting the metastasis of a sarcoma or themotility or invasive potential of a sarcoma cell, comprising evaluatingwhether or not the test substance suppresses the expression ofsarcopodin, and a substance that can be obtained by the method. In thescreening method (I) of the present invention, a substance thatsuppresses the expression of sarcopodin is selected as a substancecapable of inhibiting the metastasis of a sarcoma (particularly spindlecell sarcoma) or the motility or invasive potential of sarcoma cells(particularly spindle cell sarcoma cells).

The test substance subjected to the screening method (I) of the presentinvention may be any commonly known compound or a novel compound;examples include nucleic acids, sugars, lipids, proteins, peptides,organic low molecular compounds, compound libraries prepared usingcombinatorial chemistry technology, random peptide libraries, ornaturally occurring ingredients derived from microorganisms, animals,plants, marine organisms and the like, and the like.

The screening method (I) of the present invention comprises thefollowing steps of:

(I) bringing into contact with each other a test substance and a cellthat permits a measurement of the expression of any polypeptide orpolynucleotide selected from among (1) to (5) below;

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2,

(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell,

(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell,

(4) a polynucleotide comprising a coding region in the nucleotidesequence depicted in SEQ ID NO:1, and

(5) a polynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above;

(II) measuring the amount of any polypeptide or polynucleotide selectedfrom among (1) to (5) above expressed in the cell contacted with thetest substance, and comparing this amount expressed with the amount ofthe above-described polypeptide or polynucleotide expressed in a controlcell not contacted with the test substance; and(III) selecting a test substance that suppresses any polypeptide orpolynucleotide selected from among (1) to (5) below as a substancecapable of inhibiting the metastasis of a sarcoma or the motility orinvasive potential of a sarcoma cell on the basis of the results of thecomparison in (II) above.

The expression of any polypeptide or polynucleotide selected from among(1) to (5) accordingly means the expression of a polypeptide orpolynucleotide (preferably mRNA) of sarcopodin. The definitions for thepolypeptides or polynucleotides (1) to (5) are the same as thosedescribed in the (1. Method of determining the risk of distantmetastasis of tumor or the prognosis for survival of tumor patient)section.

“A cell that permits a measurement of the expression” refers to a cellthat permits a direct or indirect evaluation of the expression level ofan mRNA or protein which is the subject of measurement. As a cell thatpermits a direct evaluation of the expression level of an mRNA orprotein which is the subject of measurement, a cell capable ofexpressing the subject of measurement in nature can be mentioned; as acell that permits an indirect evaluation of the expression level of anmRNA or protein which is the subject of measurement, a cell that permitsa reporter assay for a transcriptional regulatory region of the subjectof measurement can be mentioned.

The cell capable of expressing the subject of measurement, that is,sarcopodin, in nature is not particularly limited, as far as itpotentially expresses the polypeptides or polynucleotides (1) to (5)above; as the cell, a primary culture cell, a cell line induced from theprimary culture cell and the like can be used. As examples of cellscapable of expressing sarcopodin in nature, sarcoma cells and the likecan be mentioned.

The cell that permits a reporter assay for a transcriptional regulatoryregion of the subject of measurement, i.e., sarcopodin, is a cellcomprising a transcriptional regulatory region (for example, a DNAconsisting of the base sequence of about 2 kbp upstream of thetranscription initiation point) of the sarcopodin gene and a reportergene (for example, GFP gene) operably linked to the region. A sarcomacell is preferred as the cell which is the subject of measurementbecause the cell expresses a physiological transcriptional regulatoryfactor for sarcopodin and is thought to be more appropriate for anevaluation of the regulation of the expression of sarcopodin.

The contact of a test substance and a cell that permits a measurement ofthe expression of sarcopodin is performed in a culture medium. A culturemedium is chosen as appropriate according to the cell that permits ameasurement of the expression of sarcopodin; examples include minimalessential medium (MEM), Dulbecco's modified essential medium (DMEM), andthe like containing about 5 to 20% fetal bovine serum. Cultivationconditions are also determined as appropriate; for example, the pH ofthe medium is about 6 to about 8, cultivation temperature is normallyabout 30 to about 40° C., and cultivation time is about 12 to about 72hours.

When a cell capable of expressing sarcopodin is used, a measurement ofthe amount of sarcopodin (polypeptides or polynucleotides (1) to (5))expressed can be performed according to the method described in the (1.Method of determining the risk of distant metastasis of tumor or theprognosis for survival of tumor patient) section. When a cell containinga reporter gene is used, the amount expressed is measured on the basisof the signal intensity of the reporter gene.

The comparison of the amounts expressed can be made preferably on thebasis of the presence or absence of a significant difference. Althoughthe amount of sarcopodin expressed in the control cell not contactedwith the test substance may be an amount expressed measured before orsimultaneously with the measurement of the amount of sarcopodinexpressed in the cell contacted with the test substance, it ispreferable, from the viewpoint of experimental accuracy andreproducibility, that the former amount expressed be a simultaneouslymeasured amount expressed.

Then, a substance that suppresses the amount of sarcopodin expressed,obtained as a result of the comparison, is selected as a substancecapable of inhibiting the metastasis of a sarcoma or the motility orinvasive potential of sarcoma cells.

A compound obtained by the screening method (I) of the present inventionis useful as a candidate substance for the development of a novelinhibitory agent for the metastasis of a sarcoma.

6. Inhibitor of Sarcoma Metastasis or Sarcoma Cell Motility

As shown in Examples below, by introducing the shorter form ofsarcopodin into a sarcoma cell expressing the longer form thereof, themotility of this sarcoma cell can be inhibited. Therefore, theshorter-form polypeptide of sarcopodin and an expression vector capableof expressing the same are useful as agents for inhibiting sarcomametastasis.

Accordingly, the present invention provides an agent for inhibiting themetastasis of a sarcoma or the motility of a sarcoma cell, containingany polypeptide selected from among (1) to (3) below or an expressionvector capable of expressing the polypeptide (inhibitor (II) of thepresent invention):

(1) a polypeptide comprising the amino acid sequence depicted in SEQ IDNO:4;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2 when expressed in the sarcoma cell; and(3) a polypeptide comprising an amino acid sequence resulting fromdeletion, substitution, insertion or addition of one or a plurality ofamino acids in the amino acid sequence depicted in SEQ ID NO:4, andpossessing an activity to inhibit the motility of a sarcoma cellexpressing the amino acid sequence depicted in SEQ ID NO:2 whenexpressed in the sarcoma cell.

The shorter-form sarcopodin polypeptide used in the inhibitor (II) ofthe present invention possesses an activity to inhibit the motility of asarcoma cell expressing a polypeptide comprising the amino acid sequencedepicted in SEQ ID NO:2 when expressed in the sarcoma cell. The activitycan be the inhibition of the cell motility enhancing activity of apolypeptide comprising the amino acid sequence depicted in SEQ ID NO:2(longer-form sarcopodin). The presence or absence of such an activity ofshorter-form sarcopodin can be determined by introducing an expressionvector capable of expressing a desired polypeptide into a sarcoma cellexpressing longer-form sarcopodin, and comparing the motility of thecell obtained with the motility of a control sarcoma cell.

The amino acid sequence contained in the polypeptide (2) above is anamino acid sequence having an identity of 90% or more, preferably 95% ormore, more preferably 98% or more, still more preferably 99% or more, tothe amino acid sequence depicted in SEQ ID NO:4. The definition for“identity” is the same as that described in the (1. Method ofdetermining the risk of distant metastasis of tumor or the prognosis forsurvival of tumor patient) section.

The amino acid sequence contained in the polypeptide (3) above is anamino acid sequence resulting from deletion, substitution, insertion oraddition of one or a plurality of amino acids in the amino acid sequencedepicted in SEQ ID NO:4, for example, (1) an amino acid sequenceresulting from deletion of one or a plurality of (preferably 1 to 30,more preferably 1 to 10, still more preferably one to several (2 to 5))amino acids in the amino acid sequence depicted in SEQ ID NO:4, (2) anamino acid sequence resulting from addition of one or a plurality of(preferably 1 to 30, more preferably 1 to 10, still more preferably oneto several (2 to 5)) amino acids in the amino acid sequence depicted inSEQ ID NO:4, (3) an amino acid sequence resulting from insertion of oneor a plurality of (preferably 1 to 30, more preferably 1 to 10, stillmore preferably one to several (2 to 5)) amino acids in the amino acidsequence depicted in SEQ ID NO:4, (4) an amino acid sequence resultingfrom substitution of one or a plurality of (preferably 1 to 30, morepreferably 1 to 10, still more preferably one to several (2 to 5)) aminoacids in the amino acid sequence depicted in SEQ ID NO:4 by other aminoacids, or (5) an amino acid sequence being a combination of themutations (1) to (4) above (in this case, the total number of mutatedamino acids is preferably 1 to 30, more preferably 1 to 10, still morepreferably one to several (2 to 5)).

In a preferred embodiment, the polypeptides (1) to (3) above are naturalshorter-form human sarcopodin polypeptides. “Natural” refers to theexistence of an amino acid sequence that constitutes a polypeptide innature. Natural shorter-form human sarcopodin amino acid sequencesinclude, but are not limited to, the amino acid sequence depicted in SEQID NO:4. It is known that a polymorphism (individual differences)usually exists in gene-constituting nucleotide sequences. Polypeptideshaving an amino acid sequence different from the amino acid sequencedepicted in SEQ ID NO:4, resulting from such polymorphism of thesarcopodin gene, are also included in natural shorter-form humansarcopodin polypeptides, as far as they possess an activity to suppressthe motility of a sarcoma cell when expressed in the sarcoma cell.

The polypeptides (1) to (3) above may comprise a tag for facilitatingprotein detection, purification and the like, and an additional sequencefor facilitating protein introduction into a cell, such as a ProteinTransduction Domain (PTD). More specifically, as the tag, a Flag tag,histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescentprotein tag (for example, GFP, YFP, RFP, CFP, BFP and the like),immunoglobulin Fc tag and the like can be mentioned. As the PTD,transcellular domains such as ANTENNAPEDIA, HIV/TAT, and HSV/VP-22, 7 to11 polyarginines and the like can be mentioned. Although the position ofthe additional sequence is not particularly limited, as far as theactivity of the polypeptides (1) to (3) above to inhibit the motility ofsarcoma cells is not affected, the position is preferably a terminal(N-terminal or C-terminal) of the polypeptide.

An expression vector capable of expressing any polypeptide selected fromamong (1) to (3) above can also be an active ingredient for theinhibitor (II) of the present invention. In the expression vector,usually, a polynucleotide (preferably DNA) comprising a nucleotidesequence that encodes the polypeptide described above (for example, thenucleotide sequence depicted in SEQ ID NO:3) is operably linked to apromoter capable of exhibiting promoter activity in cells (for example,sarcoma cells) of a mammal (preferably human) which is the subject ofadministration.

As the promoter used in the present invention, those mentioned in the(4. Agent for inhibiting the metastasis of sarcoma or the motility orinvasive potential of sarcoma cells) section can be mentioned.

The expression vector preferably comprises a transcription terminationsignal, i.e., a terminator region, downstream of a polynucleotidecomprising a nucleotide sequence that encodes the above-describedpolypeptide. Furthermore, a selection marker gene for selection oftransformant cells (a gene that confers resistance to a drug such astetracycline, ampicillin, or kanamycin, a gene that compensates for anauxotrophic mutation, and the like) can further be contained.

As the vector used in the present invention, those mentioned in the (4.Agent for inhibiting the metastasis of sarcoma or the motility orinvasive potential of sarcoma cells) section can be mentioned.

The inhibitor (II) of the present invention can comprise, in addition tothe above-described polypeptide or an expression vector capable ofexpressing the same, an optionally chosen carrier, for example, apharmaceutically acceptable carrier, and can be prepared as a medicinepreparation in the same manner as with the inhibitor (I) of the presentinvention.

The content of the above-described polypeptide or an expression vectorcapable of expressing the same in the pharmaceutical composition is, forexample, about 0.1 to 100% by weight of the entire pharmaceuticalcomposition.

Although the dosage of the inhibitor (II) of the present inventionvaries depending on the activity and choice of the active ingredient,seriousness of illness, recipient animal species, the recipient's drugreceptivity, body weight, age, and the like, and cannot be generalized,the dosage is normally about 0.001 to about 500 mg/kg, based on theamount of active ingredient, per day for an adult.

The inhibitor (II) of the present invention is preferably administeredto a mammal (for example, rat, mouse, guinea pig, rabbit, sheep, horse,swine, bovine, monkey, human) in a way such that the above-describedpolypeptide or an expression vector capable of expressing the same,which is an active ingredient thereof, is delivered to a sarcoma tissue(sarcoma cells).

Since the above-described polypeptide possesses an activity to inhibitthe motility or invasive potential of a sarcoma cell, metastasis ofsarcomas (particularly spindle cell sarcoma) can be inhibited to improvethe prognosis for survival by administering the inhibitor (II) of thepresent invention to a patient with a sarcoma (particularly spindle cellsarcoma) or a patient after treatment of a sarcoma at a risk of distantmetastasis of the sarcoma, and the like.

7. Screening Method (II) for Substance Capable of Inhibiting SarcomaMetastasis and the Like

As stated above, by introducing the shorter-form of sarcopodin into asarcoma cell expressing the longer form thereof, the motility of thissarcoma cell can be inhibited. This finding shows that by inhibiting afunction of longer-form sarcopodin using an appropriately designeddeletion mutant sarcopodin (partial peptide), it is possible to inhibitthe motility of a sarcoma cell and hence inhibit the metastasis of asarcoma. As shown in Examples, longer-form sarcopodin binds to actin andSrc. Therefore, the deletion mutant is possibly one capable ofinhibiting this binding, but this mode of inhibition is not to beconstrued as limiting.

On the basis of these findings, the present invention provides a methodof screening for a polypeptide capable of inhibiting the metastasis of asarcoma or the motility of sarcoma cells, comprising the following stepsof:

(I) expressing a test polypeptide comprising at least three consecutiveamino acids contained in the amino acid sequence depicted in SEQ ID NO:2in a sarcoma cell expressing a polypeptide comprising the amino acidsequence depicted in SEQ ID NO:2;(II) measuring the motility of the cell in which the test polypeptide isexpressed, and comparing this motility with the motility of a controlsarcoma cell in which the test polypeptide is not expressed;(III) selecting a polypeptide that has suppressed the motility of thesarcoma cell as a polypeptide capable of inhibiting the metastasis of asarcoma or the motility of sarcoma cells on the basis of the results ofthe comparison in (II) above, and a polypeptide that can be obtained bythe method.

The test polypeptide subjected to the screening method (II) of thepresent invention comprises a partial sequence consisting of at leastthree consecutive amino acids contained in the amino acid sequencedepicted in SEQ ID NO:2. The length of the partial sequence is notparticularly limited; however, to achieve a sufficient inhibitoryactivity, the length is preferably 5 amino acids or more, morepreferably 10 amino acids or more.

Because the motility of a sarcoma cell can be inhibited by introductionof shorter-form sarcopodin, a polypeptide that potently inhibits themotility of a sarcoma cell can be obtained at a higher probability bydesigning a test polypeptide on the basis of this shorter-formsarcopodin. Therefore, in a preferred embodiment, the test polypeptidesubjected to the screening method (II) of the present inventioncomprises a partial sequence consisting of at least three consecutiveamino acids contained in the amino acid sequence depicted in SEQ IDNO:4.

The expression of a test polypeptide in a sarcoma cell can be achievedby introducing an expression vector capable of expressing the testpolypeptide into the sarcoma cell. In the expression vector, apolynucleotide comprising a nucleotide sequence that encodes the testpolypeptide is operably linked to a promoter capable of exhibitingpromoter activity in the sarcoma cell.

As the promoter used, those mentioned in the (4. Agent for inhibitingthe metastasis of sarcoma or the motility or invasive potential ofsarcoma cells) section can be mentioned.

The expression vector preferably comprises a transcription terminationsignal, i.e., a terminator region, downstream of a polynucleotidecomprising a nucleotide sequence that encodes the test polypeptide.Furthermore, a selection marker gene for selection of transformant cells(a gene that confers resistance to a drug such as tetracycline,ampicillin, or kanamycin, a gene that compensates for an auxotrophicmutation, and the like) can further be contained.

As the vector used in the present invention, those mentioned in the (4.Agent for inhibiting the metastasis of sarcoma or the motility orinvasive potential of sarcoma cells) section can be mentioned.

Introduction of an expression vector into a sarcoma cell can beperformed by an ordinary method of transfection, such as the calciumphosphate method, DEAE dextran method, electroporation, or lipofection.When a virus is used as the vector, the genome of the virus may beintroduced into the cell by one of the ordinary method of transfectiondescribed above, and the genome of the virus can also be introduced intothe cell by infecting the cell with a virion.

The sarcoma cell used in a method of the present invention is normallyderived from a mammal, preferably derived from a human. The sarcoma cellis expressing a polypeptide comprising the amino acid sequence depictedin SEQ ID NO:2 (longer-form human sarcopodin) naturally or forcibly.Forced expression of the polypeptide can be achieved by introducing anexpression vector capable of expressing the polypeptide, as describedabove.

The motility of a cell in which the test polypeptide is expressed can bemeasured according to, for example, the gold colloid method, Boydenchamber assay and the like.

Then, the motility of the cell in which the test polypeptide isexpressed is compared with the motility of a control sarcoma cell inwhich the test polypeptide is not expressed.

The comparison of the motilities can be made preferably on the basis ofthe presence or absence of a significant difference. Although themotility of the control cell in which the test polypeptide is notexpressed may be measured before or simultaneously with the measurementof the motility of the cell in which the test polypeptide is expressed,it is preferable, from the viewpoint of experimental accuracy andreproducibility, that the former motility be a simultaneously measuredvalue.

Then, a polypeptide that has suppressed the motility of the sarcomacell, obtained as a result of the comparison, is selected as apolypeptide capable of inhibiting the metastasis of a sarcoma or themotility of sarcoma cells.

A polypeptide obtained by the screening method (II) of the presentinvention is useful as a candidate substance for the development of anovel inhibitory agent for sarcoma metastasis.

The present invention is hereinafter described in more detail by meansof the following Examples, to which, however, the present invention isnot limited in any way.

EXAMPLES Example 1 Expression of Sarcopodin in Spindle Cell Sarcoma andFunctional Analysis Thereof Identification of Sarcopodin Gene

In the Examples below, the longer form of sarcopodin is referred to asC7059, and the shorter form of sarcopodin as ctC7059.

cDNAs were prepared from RNAs extracted from spindle cell sarcomatissues of 65 patients, and a comprehensive gene expression analysis wasperformed using a cDNA microarray on about 23000 kinds of genes preparedat the Institute of Medical Science in the University of Tokyo (FIG. 1).As a reference control, a cDNA prepared from an RNA extracted frommesenchymal stem cells (hereinafter MSCs) was used. The 65 patients weredivided into two groups according to the presence or absence of onset ofdistant metastasis or tumor-related death, and they were examined forgenes exhibiting a statistically significant difference in theexpression level between the two groups. As a result, C7059 wasidentified as a gene exhibiting the strongest correlation with both theonset of distant metastasis and tumor-related death (FIG. 2).

On the basis of the pathologic findings, spindle cell sarcoma patientswere classified into some groups; irrespective of the pathologicclassification, the expression level of C7059 was high in patientspositive for distant metastasis (FIG. 3).

For factors other than the amount of gene expressed as well, thecorrelations with tumor-related death in spindle cell sarcoma wereanalyzed. However, of the factors analyzed, only the C7059 expressionlevel exhibited a significant correlation with tumor-related death (FIG.4).

These results suggested that by measuring the expression level of C7059in spindle cell sarcoma tissue, the risk of onset of distant metastasisor the prognosis for survival of the patient could be determined.

Analysis of Information on C7059

C7059 was identified as a functionally unknown gene registered withGenBank as AK074185 (SEQ ID NO:1). C7059 is a gene present in the longarm region of chromosome 5 (5q32), consisting of 19 exons; the cDNA andORF thereof were found to have lengths of 4153 bp and 2304 bp,respectively, and encode a protein configured with 768 amino acids(accession number: FLJ00258) (SEQ ID NO:2) (FIG. 5). This gene also havea transcription initiation point within the 9th intron, the shorter formof C7059 (referred to as ctC7059), having the same structure as theC-terminal of C7059, was found to be registered as AK094067 (SEQ IDNO:3) (FIGS. 6 and 7).

A portion of a DNA that encodes C7059 was amplified by PCR using thefollowing primers, and the portion was used as a probe for cDNAmicroarray.

Forward: tggtggtgcagtgtaaagagacaaga (SEQ ID NO: 1 nucleotide numbers2786 to 2811: SEQ ID NO: 5) Reverse: gtttagcacccatgtgacagaa (SEQ ID NO:1 nucleotide numbers 3355 to 3334: SEQ ID NO: 6)

This probe hybridizes to a common region shared by C7059 and ctC7059.Hence, the sum of the amounts of C7059 and ctC7059 expressed isdetected.

It was also found by quantitative RT-PCR that the expression of ctC7059in spindle cell sarcoma was lower than the expression of C7059 (FIG. 8).From these findings, it was estimated that the amount of C7059 expressedmainly correlates with the onset of distant metastasis and tumor-relateddeath.

The nucleotide sequences of the primers used in the above-describedRT-PCR are as follows:

Primers for C7059 amplification Forward: atcggcagccacatctgaggttg (SEQ IDNO: 1 nucleotide numbers 841 to 863: SEQ ID NO: 7) Reverse:ttgtcacccacacccacgct (SEQ ID NO: 1 nucleotide numbers 1150 to 1131: SEQID NO: 8) Primers for ctC7059 amplification Forward:gtatggtatgaggcacaggatag (SEQ ID NO: 3 nucleotide numbers 30 to 52: SEQID NO: 9) Reverse: gcgttcacatgggttcgcag (SEQ ID NO: 1 nucleotide numbers1453 to 1434: SEQ ID NO: 10)

This finding suggested that the risk of the onset of distant metastasisof spindle cell sarcoma or the prognosis for survival of the patientcould be determined by specifically detecting C7059.

Verification of Microarray Data

Probes for quantitative RT-PCR were generated from the genome sequenceof C7059, and the expression of C7059 in the spindle cell sarcoma tissueused in the microarray was quantitatively analyzed by the TaqMan method.

The nucleotide sequences of the primers and probe used in the TaqManmethod are as follows:

Forward: aacaagcctccccctgaggact (SEQ ID NO: 1 nucleotide numbers 429 to450: SEQ ID NO: 11) Reverse: gttgcagggtagctgctgtc (SEQ ID NO: 1nucleotide numbers 580 to 561: SEQ ID NO: 12) TaqMan probe:ggcccttcctctgggacccggc (SEQ ID NO: 1 nucleotide numbers 461 to 482: SEQID NO: 13)

Because these primers are specific for C7059 and do not detect ctC7059,the amount of only C7059 expressed can be measured quantitatively.

As a result, the results of the quantitative RT-PCR generally agreedwith the microarray data (FIGS. 8A and B); with the expression level ofreference control mesenchymal stem cells taken as 1, the patients weredivided into two groups: a group having an expression level of 10 ormore and a group having an expression level of less than 10; theincidence of distant metastasis were compared; in the group with anexpression level of 10 or more, distant metastasis occurred at a higherrate with a statistically significant difference (FIGS. 9A and B).Furthermore, a similar analysis was performed using the results from 10newly enrolled patients; as expected, in patients with high expressionof C7059, distant metastasis occurred at a higher frequency (FIGS. 9Aand C).

Preparation of Specific Antibody

Domestic rabbits were immunized with a fusion protein of anamino-terminal region of C7059 (the region shown by amino acid numbers35 to 113 in SEQ ID NO:2) and GST as an antigen to generate aC7059-specific polyclonal antibody (FIG. 10A). By Western blot using theantibody obtained, C7059 was found to be an about 110 kD protein presentmainly in the cytoplasm (FIG. 10B).

Immunohistochemical Staining

The C7059 expression vector described below was introduced intoC7059-negative sarcoma cells (SaOS2). The cells were stained using theanti-C7059 antibody described above. As a result, C7059 was found tobind with actin and aggregate particularly in the lamellipodia (FIG.11).

Formalin-fixed tumor tissues collected from spindle cell sarcomapatients were stained using the anti-C7059 antibody described above. Asa result, C7059 stainability was observed mainly in the cytoplasm, andpartially in the nucleus. Agreeing with the analytical results at theRNA level, the sarcoma tissue from the patient positive for distantmetastasis and positive for tumor-related death was strongly positivefor C7059, and the sarcoma tissue from the patient negative for distantmetastasis and negative for tumor-related death was weakly positive forC7059 (FIG. 12). This result showed that the risk of onset of distantmetastasis in spindle cell sarcoma and the prognosis for survival of thepatient could also be determined by immunohistochemical staining using atissue section.

The sarcoma tissue from the patient positive for distant metastasis andpositive for tumor-related death exhibited strong C7059 expression inthe cytoplasm and nucleus, whereas the sarcoma tissue from the patientnegative for distant metastasis and negative for tumor-related deathexhibited weak C7059 expression in the nucleus only (FIG. 12). Thisresult suggested that the intracellular localization of the C7059protein might vary depending on the malignancy of sarcoma cells.

Effects of Induction or Inhibition of C7059 Expression on Cell Motilityand Invasive Potential

By inserting a DNA that encodes C7059 into a multicloning site ofpCAGGS, a vector capable of transiently strongly expressing the C7059protein was prepared (FIG. 13). The C7059 expression vector wasintroduced into a C7059-negative cultured sarcoma cell (SaOS2), and themotility and invasive potential of the cell were evaluated.

The motility and invasive potential of the cell were evaluated by Boydenchamber assay. 2.5×10⁴ of the cells were seeded to a 24-well platehaving a PET membrane with 8-micron pores on the bottom thereof; 22hours later, the cells that had passed the membrane were counted toobtain an index of cell motility. Next, using a porous PET membranecoated with a synthetic basal membrane tissue, the same experiment wasperformed; the cells that destroyed the basal membrane tissue andinvaded were counted. Using the quota obtained by dividing the number ofinvasive cells by the number of migrating cells, the cell invasivepotential was evaluated.

As a result, with the introduction of the C7059 expression vector, thesarcoma cell motility did not change, but the invasive potential wassignificantly enhanced (FIG. 14A). This result suggested that anelevation of C7059 expression in spindle cell sarcoma might cause anenhancement of the invasive potential, which in turn can result in anincreased incidence of distant metastasis and a worse prognosis forsurvival of the patient.

The following 4 kinds of siRNAs against C7059 were designed:

C7059 siRNA1-1: cagggugaacggcgagcuuaa (SEQ ID NO: 1 nucleotide numbers581 to 601: SEQ ID NO: 14) C7059 siRNA1-2: cagcaucaucuacgugcccaa (SEQ IDNO:1 nucleotide numbers 878 to 898: SEQ ID NO: 15) C7059 siRNA1-3:aucccucuuugaagaauuuga (SEQ ID NO: 1 nucleotide numbers 284 to 304: SEQID NO: 16) C7059 siRNA1-4: aaaggaggucuccuaccugua (SEQ ID NO: 1nucleotide numbers 221 to 241: SEQ ID NO: 17)

Double-stranded RNAs were prepared from these siRNAs and introduced intoC7059-positive cultured sarcoma cells (U2OS); suppression of C7059expression at the mRNA level and protein level was confirmed (FIG. 13B).Of these RNAs, that having the most potent suppressive effect, C7059siRNA1-3, was used in the following assay.

C7059 siRNA1-3 was introduced into a C7059-positive cultured sarcomacell, and the motility and invasive potential of the cell wereevaluated.

As a result, it was found that by inhibition of C7059 expression by thesiRNA, the motility and invasive potential of the sarcoma cell wereremarkably inhibited (FIG. 14B). This result suggested that bysuppressing C7059 expression in sarcoma cells, the metastasis of asarcoma might be inhibited to improve the prognosis for survival of thepatient.

Establishment of Immortalized MSCs that Stably Express C7059 UsingLentivirus Vector

A lentivirus vector capable of strongly expressing the C7059 protein wasconstructed.

The region of nucleotide numbers 75 to 2381 in SEQ ID NO:1 was amplifiedby PCR and inserted into the cloning vector pENTR/D/TOPO by TOPOcloning, whereby pENTR/C7059 was prepared.

Subsequently, pENTR/C7059 and pLenti6/V5-DEST were LR-recombined, andpLenti/C7059 was prepared by double selection with ampicillin andblasticidin.

As described in BBRC, vol. 353, p. 60-66 (2007) and J Gene Med, vol. 6,p. 833-845 (2004), the Bmi1 gene and the human TERT gene were introducedinto human MSCs (mesenchymal stem cells) purchased from BIOWHITTAKERCo., using a retrovirus, and chemical selection (G418 100 μg/ml, orhygromycin B 50 μg/ml) was performed, whereby an immortalized parent MSCline was acquired.

The C7059 expression lentivirus vector was introduced into theimmortalized MSCs to establish an immortalized MSC line that expressesC7059 stably, and the motility, invasive potential andanchorage-independent growth of the cells were evaluated.

The anchorage-independent growth was evaluated by the method describedbelow. A double agar medium comprising 0.7% of agar in the lower layerand 0.35% of agar in the upper layer was prepared using a 60 mm dish;1×10⁴ cells were seeded to the upper layer. After cultivation for 4weeks, the colonies formed were stained and counted.

As a result, with the introduction of the C7059 expression vector, themotility, invasive potential and anchorage-independent growth potentialof the MSCs were significantly enhanced (FIG. 15). This result suggestedthat induction of the expression of C7059 might transform immortalizedMSCs cancerous.

Binding of C7059 and Src

By inserting a DNA that encodes a polypeptide comprising C7059 and threeFlag tags linked to the N-terminus thereof (3× Flag-c7059) into amulticloning site of pCAGGS, a vector capable of transiently stronglyexpressing the 3× Flag-c7059 protein was prepared. pCAGGS-3× Flag-c7059was introduced into C7059-negative sarcoma cells (SaOS2), and the cellswere analyzed for the presence or absence of an interaction betweenC7059 and Src by immunoprecipitation.

When cells were immunoprecipitated with anti-Flag antibody and blottedwith anti-Src antibody, a specific band was identified only in the cellstransfected with pCAGGS-3× Flag-c7059 (FIG. 16(2)). Even when cells wereimmunoprecipitated with the anti-Src antibody and blotted with theanti-C7059 antibody or anti-Flag antibody, a specific band was foundonly in the cells transfected with pCAGGS-3× Flag-c7059 (FIGS. 16(3) and(4)).

These results demonstrated that C7059 has the capability of binding toSrc. Immunocytological staining also showed that C7059 was co-localizedwith Src and aggregated intensely particularly in the lamellipodia (FIG.17).

By inserting a DNA that encodes a polypeptide comprising ctC7059 andthree Flag tags linked to the N-terminus thereof (3FH-ctC7059) into amulticloning site of pCAGGS, a vector capable of transiently stronglyexpressing the 3FH-ctC7059 protein was prepared (FIG. 18A). The3FH-ctC7059 expression vector was introduced into a C7059 stronglypositive sarcoma cell line (U2OS), and the motility of the cells wasevaluated.

As a result, with introduction of the ctC7059 expression vector, themotility of the sarcoma cells decreased significantly (FIG. 18B). Thisresult suggested that ctC7059 might possess antagonistic action on C7059and possess an activity to inhibit the metastasis of a sarcoma.

Example 2 Expression of Sarcopodin in Colorectal Cancer (Methods)

The subjects were 39 patients with colic or rectal cancer who underwentsurgery at Kyoto University Hospital between 1999 and the present.Separated tumor tissues were fixed with formalin, and subjected toimmunohistochemical staining using the anti-C7059 antibody prepared inExample 1. A non-tumoral portion adjacent to the tumor tissue was usedas a normal mucosa.

(Results)

In the normal mucosa, the expression of the C7059 protein was littleobserved (FIGS. 19 to 23). Expression localized in the nucleus wasrarely observed.

In the polyps as well, the expression of the C7059 protein was notobserved, or the expression of the C7059 protein localized in thenucleus was observed. No expression in the cytoplasm was observed.

In contrast, in tumor cells, the expression of the C7059 protein wasobserved in many patients (26/39) (FIGS. 19 to 23). Although theexpression in the cytoplasm was predominant, expression was alsoobserved in the nucleus. Results of an evaluation of the stainability inthe cytoplasm in four grades (no stain, weakly positive, positive,strongly positive) are as follows:

No stain: 13 (33.3%)Weakly positive: 12 (30.1%)

Positive: 12 (30.1%)

Strongly positive: 2 (0.5%)

These results suggested that C7059 is a molecule whose expressionincreases with the tumorization of a colorectal mucosal cell, and thatby evaluating the presence or absence of the expression of C7059,whether or not the colorectal mucosal cell is a tumor cell can bedetermined. It was also suggested that by evaluating whether the C7059expression site in the cell is the cytoplasm or is localized in thenucleus, tumors and polyps in the large intestine could be distinguishedfrom each other.

At tumor sites of low heterogeneity, C7059 expression in the nucleus wasdistinctive; at tumor sites of high heterogeneity, such as invasivefronts, however, C7059 expression in the cytoplasm was predominant (FIG.23). This finding showed that in colorectal cancer, like in spindle cellsarcoma, the localization of the C7059 protein possibly varies dependingon the malignancy of the cell.

Additionally, judging from these findings combined with clinicalinformation, the positive patients were identified as patients in thepoor prognosis group (Stage 1V) or those experiencing a local recurrenceor metastasis.

This finding suggested that by evaluating the expression level of C7059,the risk of metastasis and the prognosis for survival in colorectalcancer could be determined.

Example 3 Expression of Sarcopodin in Colorectal Cancer (2)

The subjects were 15 patients with colic or rectal cancer who underwentsurgery at Kyoto University Hospital between 2006 and 2007. Separatednormal tissue and tumor tissue of the colon-rectum were stored underfreezing, and C7059 expression was measured by quantitative RT-PCR inthe same manner as Example 1. As a result, in 10 of the 15 patients,C7059 expression was enhanced in the colic or rectal tumor tissue,compared with the normal colic or rectal tissue (FIG. 24).

This result suggested that C7059 is a molecule whose expressionincreases with the tumorization of a colorectal mucosal cell, and thatby evaluating the presence or absence of the expression of C7059,whether or not the colorectal mucosal cell is a tumor cell can bedetermined.

Example 4 Expression of Sarcopodin in Colorectal Cancer (3) (Methods)

The subjects were 164 patients with colic or rectal cancer who underwentsurgery at Kyoto University Hospital between 1999 and 2001. Separatedtumor tissue was fixed with formalin, and subjected toimmunohistochemical staining using the anti-C7059 antibody prepared inExample 1. A non-tumoral portion adjacent to the tumor tissue was usedas a normal mucosa.

(Results)

In tumor cells, in many patients, expression of the C7059 protein wasobserved (124/164). Results of an evaluation of the stainability in thecytoplasm in three grades (no stain, weakly positive, strongly positive)are as follows:

No stain: 40 (24.4%)Weakly positive: 53 (32.3%)Strongly positive: 71 (43.3%)

These results showed that C7059 is a molecule whose expression increaseswith the tumorization of a colorectal mucosal cell, and that byevaluating the presence or absence of the expression of C7059, whetheror not the colorectal mucosal cell is a tumor cell can be determined.

Stage III patients with strong expression of C7059 (21/52) tended to bemore likely to experience a local recurrence or metastasis than patientsin the same stage without expression of C7059 (9/52) (FIG. 25).

In rectal cancer patients undergoing radical surgery to resect a tumorsite (80 patients), patients with high expression of C7059 tended to bemore likely to experience a local recurrence or metastasis than patientswithout expression of C7059 (FIG. 26).

The results above showed that by evaluating the expression level ofC7059, the risk of recurrence/metastasis and prognosis for survival incolorectal cancer can be determined.

INDUSTRIAL APPLICABILITY

Using the method of the present invention, it is possible to quicklydetermine the risk of onset of distant metastasis or prognosis forsurvival in a tumor such as spindle cell sarcoma or colorectal cancerusing a convenient technique such as immunological staining using anantibody or quantitative RT-PCR using a specific probe. Using the methodof the present invention, it is possible to administer a selectivetreatment to the patient on the basis of the level of the risk of onsetof distant metastasis or the determination of prognosis for survival,thus improving the quality of tumor treatment. Specifically, forpatients judged to have a poor prognosis using the method of the presentinvention, an improvement of the prognosis can be aimed at byproactively performing an adjuvant therapy, and attempting for earlydetection of metastatic foci by regular diagnostic imaging. Conversely,for patients judged to have a good prognosis using the method of thepresent invention, unnecessary adjuvant therapy can be avoided. Inparticular, because the method of the present invention is applicable toall types of spindle cell sarcoma, the prognosis for survival can bedetermined accurately even if a pathologic diagnosis is unestablished.

Using the method of the present invention, the presence or absence ofcolorectal cancer cells and the malignancy of colorectal cancer cellscan be determined with high accuracy.

Using the agent for inhibiting metastasis of the present invention, itis possible to selectively suppress the metastasis of spindle cellsarcoma and improve the prognosis for survival in the spindle cellsarcoma. The agent for inhibiting metastasis of the present invention isdistinctive in that the onset of distant metastasis, which is a factorthat determines the prognosis of spindle cell sarcoma, is targeted.Because the expression levels in normal tissue are much lower than thosein tumor tissue, the adverse reactions caused by expression suppressingtreatment with an siRNA are expected to be very mild, and the treatmentenables an effective treatment of spindle cell sarcoma when combinedwith currently available therapeutic methods.

This application is based on a patent application No. 2007-145827 filedin Japan (filing date: May 31, 2007), the contents of which areincorporated in full herein by this reference.

1. A method of determining the risk of distant metastasis of a tumor,comprising measuring the expression level of any polypeptide orpolynucleotide selected from among (1) to (5) below in tumor tissue, anddetermining the risk of distant metastasis of the tumor on the basis ofthe correlation between the expression level and the incidence ofdistant metastasis: (1) a polypeptide comprising the amino acid sequencedepicted in SEQ ID NO:2; (2) a polypeptide comprising an amino acidsequence having an identity of 90% or more to the amino acid sequencedepicted in SEQ ID NO:2, and possessing an activity to enhance themotility or invasive potential of a cell when expressed in the cell; (3)a polypeptide comprising an amino acid sequence resulting from deletion,substitution, insertion or addition of one or a plurality of amino acidsin the amino acid sequence depicted in SEQ ID NO:2, and possessing anactivity to enhance the motility or invasive potential of a cell whenexpressed in the cell; (4) a polynucleotide comprising a coding regionin the nucleotide sequence depicted in SEQ ID NO:1; and (5) apolynucleotide comprising a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) above.
 2. The methodaccording to claim 1, wherein the expression level is measured using anysubstance selected from among (i) to (iii) below: (i) an antibody thatspecifically recognizes a polypeptide consisting of the amino acidsequence depicted in SEQ ID NO:2; (ii) a nucleic acid probe or primercapable of specifically detecting a polynucleotide consisting of thenucleotide sequence depicted in SEQ ID NO:1; and (iii) a nucleic acidprobe or primer capable of specifically detecting a polynucleotideconsisting of a nucleotide sequence that encodes a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2.
 3. Themethod according to claim 2, wherein the substance used for themeasurement is any one selected from among (ia) to (iiia) below: (ia) anantibody that specifically recognizes a polypeptide consisting of theamino acid sequence depicted in SEQ ID NO:2, a recognition epitope forthe antibody being present within the region shown by amino acid numbers1 to 385 in SEQ ID NO:2; (iia) a nucleic acid probe or primer capable ofspecifically detecting a polynucleotide consisting of the nucleotidesequence shown by nucleotide numbers 1 to 1229 in SEQ ID NO:1; and(iiia) a nucleic acid probe or primer capable of specifically detectinga polynucleotide consisting of a nucleotide sequence that encodes apolypeptide consisting of the amino acid sequence shown by amino acidnumbers 1 to 385 in SEQ ID NO:2.
 4. The method according to claim 1,wherein the tumor is spindle cell sarcoma or colorectal cancer.
 5. Amethod of determining the prognosis for survival of a tumor patient,comprising measuring the expression level of any polypeptide orpolynucleotide selected from among (1) to (5) below in tumor tissue, anddetermining the prognosis for survival of the tumor patient on the basisof the correlation between the expression level and the determination ofthe prognosis for survival of the tumor patient: (1) a polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:2; (2) apolypeptide comprising an amino acid sequence having an identity of 90%or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell; (3) a polypeptide comprising an aminoacid sequence resulting from deletion, substitution, insertion oraddition of one or a plurality of amino acids in the amino acid sequencedepicted in SEQ ID NO:2, and possessing an activity to enhance themotility or invasive potential of a cell when expressed in the cell; (4)a polynucleotide comprising a coding region in the nucleotide sequencedepicted in SEQ ID NO:1; and (5) a polynucleotide comprising anucleotide sequence that encodes any polypeptide selected from among (1)to (3) above.
 6. The method according to claim 5, wherein the expressionlevel is measured using any substance selected from among (i) to (iii)below: (i) an antibody that specifically recognizes a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2; (ii) anucleic acid probe or primer capable of specifically detecting apolynucleotide consisting of the nucleotide sequence depicted in SEQ IDNO:1; and (iii) a nucleic acid probe or primer capable of specificallydetecting a polynucleotide consisting of a nucleotide sequence thatencodes a polypeptide consisting of the amino acid sequence depicted inSEQ ID NO:2.
 7. The method according to claim 6, wherein the substanceused for the measurement is any one selected from among (ia) to (iiia)below: (ia) an antibody that specifically recognizes a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2, arecognition epitope for the antibody being present within the regionshown by amino acid numbers 1 to 385 in SEQ ID NO:2; (iia) a nucleicacid probe or primer capable of specifically detecting a polynucleotideconsisting of the nucleotide sequence shown by nucleotide numbers 1 to1229 in SEQ ID NO:1; and (iiia) a nucleic acid probe or primer capableof specifically detecting a polynucleotide consisting of a nucleotidesequence that encodes a polypeptide consisting of the amino acidsequence shown by amino acid numbers 1 to 385 in SEQ ID NO:2.
 8. Themethod according to claim 5, wherein the tumor is spindle cell sarcomaor colorectal cancer. 9.-14. (canceled)
 15. A method of detectingcolorectal cancer cells in colorectal mucosal tissue, comprisingevaluating the expression of any polypeptide or polynucleotide selectedfrom among (1) to (5) below in colorectal mucosal tissue, anddetermining whether or not colorectal cancer cells are contained in thetissue on the basis of the presence or absence of the expression: (1) apolypeptide comprising the amino acid sequence depicted in SEQ ID NO:2;(2) a polypeptide comprising an amino acid sequence having an identityof 90% or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell; (3) a polypeptide comprising an aminoacid sequence resulting from deletion, substitution, insertion oraddition of one or a plurality of amino acids in the amino acid sequencedepicted in SEQ ID NO:2, and possessing an activity to enhance themotility or invasive potential of a cell when expressed in the cell; (4)a polynucleotide comprising a coding region in the nucleotide sequencedepicted in SEQ ID NO:1; and (5) a polynucleotide comprising anucleotide sequence that encodes any polypeptide selected from among (1)to (3) above.
 16. (canceled)
 17. A method of determining the malignancyof a tumor cell, comprising evaluating the localization of anypolypeptide selected from among (1) to (3) below in the tumor cell, anddetermining the malignancy of the tumor cell on the basis of thepresence or absence of the expression of the polypeptide in thecytoplasm: (1) a polypeptide comprising the amino acid sequence depictedin SEQ ID NO:2; (2) a polypeptide comprising an amino acid sequencehaving an identity of 90% or more to the amino acid sequence depicted inSEQ ID NO:2, and possessing an activity to enhance the motility orinvasive potential of a cell when expressed in the cell; and (3) apolypeptide comprising an amino acid sequence resulting from deletion,substitution, insertion or addition of one or a plurality of amino acidsin the amino acid sequence depicted in SEQ ID NO:2, and possessing anactivity to enhance the motility or invasive potential of a cell whenexpressed in the cell.
 18. The method according to claim 17, wherein thetumor is spindle cell sarcoma or colorectal cancer. 19.-22. (canceled)23. A method of screening for a substance capable of inhibiting themetastasis of a sarcoma or the motility or invasive potential of asarcoma cell, comprising the following steps of: (I) bringing intocontact with each other a test substance and a cell that permits ameasurement of the expression of any polypeptide or polynucleotideselected from among (1) to (5) below: (1) a polypeptide comprising theamino acid sequence depicted in SEQ ID NO:2, (2) a polypeptidecomprising an amino acid sequence having an identity of 90% or more tothe amino acid sequence depicted in SEQ ID NO:2, and possessing anactivity to enhance the motility or invasive potential of a cell whenexpressed in the cell, (3) a polypeptide comprising an amino acidsequence resulting from deletion, substitution, insertion or addition ofone or a plurality of amino acids in the amino acid sequence depicted inSEQ ID NO:2, and possessing an activity to enhance the motility orinvasive potential of a cell when expressed in the cell, (4) apolynucleotide comprising a coding region in the nucleotide sequencedepicted in SEQ ID NO:1, and (5) a polynucleotide comprising anucleotide sequence that encodes any polypeptide selected from among (1)to (3) above; (II) measuring the amount of any polypeptide orpolynucleotide selected from among (1) to (5) above expressed in thecell contacted with the test substance, and comparing this amountexpressed with the amount of the above-described polypeptide orpolynucleotide expressed in a control cell not contacted with the testsubstance; and (III) selecting a test substance that suppresses theamount of any polypeptide or polynucleotide selected from among (1) to(5) above expressed, as a substance capable of inhibiting the metastasisof a sarcoma or the motility or invasive potential of a sarcoma cell, onthe basis of the results of the comparison in (II) above. 24.-25.(canceled)
 26. A method of screening for a peptide capable of inhibitingthe metastasis of a sarcoma or the motility of a sarcoma cell,comprising the following steps of: (I) expressing a test polypeptidecomprising at least three consecutive amino acids contained in the aminoacid sequence depicted in SEQ ID NO:2 in a sarcoma cell expressing apolypeptide comprising the amino acid sequence depicted in SEQ ID NO:2;(II) measuring the motility of the cell in which the test polypeptidehas been expressed, and comparing this motility with the motility of acontrol sarcoma cell in which the test polypeptide has not beenexpressed; (III) selecting a polypeptide that has suppressed themotility of the sarcoma cell as a polypeptide capable of inhibiting themetastasis of a sarcoma or the motility of a sarcoma cell, on the basisof the results of the comparison in (II) above. 27.-39. (canceled)
 40. Amethod for inhibiting the metastasis of a sarcoma in a mammal,comprising administering to the mammal an effective amount of anypolynucleotide selected from among (a) to (d) below or an expressionvector capable of expressing the polynucleotide: (a) a polynucleotidecomprising a nucleotide sequence complementary to the nucleotidesequence depicted in SEQ ID NO:1, the nucleotide sequence of an initialtranscription product that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more; (b) a polynucleotide comprising a nucleotidesequence complementary to a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) below, the nucleotidesequence of an initial transcription product that produces the formernucleotide sequence by post-transcriptional processing, or a partialsequence thereof as long as 15 bases or more; (1) a polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:2, (2) apolypeptide comprising an amino acid sequence having an identity of 90%or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and (3) a polypeptide comprising anamino acid sequence resulting from deletion, substitution, insertion oraddition of one or a plurality of amino acids in the amino acid sequencedepicted in SEQ ID NO:2, and possessing an activity to enhance themotility or invasive potential of a cell when expressed in the cell; (c)a polynucleotide comprising a nucleotide sequence hybridizable, underphysiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and (d) a polynucleotide comprising a nucleotidesequence hybridizable, under physiological conditions for an animalwhich is the subject of treatment, with a polynucleotide consisting of anucleotide sequence that encodes any polypeptide selected from among (1)to (3) above, or an initial transcription product that produces theformer nucleotide sequence by post-transcriptional processing, andsuppressing the transcription or translation of any one of thepolypeptides (1) to (3) in the hybridized state.
 41. A method forinhibiting the motility or invasive potential of a sarcoma cell in amammal, comprising administering to the mammal an effective amount ofany polynucleotide selected from among (a) to (d) below or an expressionvector capable of expressing the polynucleotide: (a) a polynucleotidecomprising a nucleotide sequence complementary to the nucleotidesequence depicted in SEQ ID NO:1, the nucleotide sequence of an initialtranscription product that produces the former nucleotide sequence bypost-transcriptional processing, or a partial sequence thereof as longas 15 bases or more; (b) a polynucleotide comprising a nucleotidesequence complementary to a nucleotide sequence that encodes anypolypeptide selected from among (1) to (3) below, the nucleotidesequence of an initial transcription product that produces the formernucleotide sequence by post-transcriptional processing, or a partialsequence thereof as long as 15 bases or more; (1) a polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:2, (2) apolypeptide comprising an amino acid sequence having an identity of 90%or more to the amino acid sequence depicted in SEQ ID NO:2, andpossessing an activity to enhance the motility or invasive potential ofa cell when expressed in the cell, and (3) a polypeptide comprising anamino acid sequence resulting from deletion, substitution, insertion oraddition of one or a plurality of amino acids in the amino acid sequencedepicted in SEQ ID NO:2, and possessing an activity to enhance themotility or invasive potential of a cell when expressed in the cell; (c)a polynucleotide comprising a nucleotide sequence hybridizable, underphysiological conditions for an animal which is the subject oftreatment, with a polynucleotide consisting of the nucleotide sequencedepicted in SEQ ID NO:1, or an initial transcription product thatproduces the former polynucleotide by post-transcriptional processing,and suppressing the transcription or translation of a polypeptideconsisting of the amino acid sequence depicted in SEQ ID NO:2 in thehybridized state; and (d) a polynucleotide comprising a nucleotidesequence hybridizable, under physiological conditions for an animalwhich is the subject of treatment, with a polynucleotide consisting of anucleotide sequence that encodes any polypeptide selected from among (1)to (3) above, or an initial transcription product that produces theformer nucleotide sequence by post-transcriptional processing, andsuppressing the transcription or translation of any one of thepolypeptides (1) to (3) in the hybridized state.
 42. A method forinhibiting the metastasis of a sarcoma in a mammal, comprisingadministering to the mammal an effective amount of any polypeptideselected from among (1) to (3) below or an expression vector capable ofexpressing the polypeptide: (1) a polypeptide comprising the amino acidsequence depicted in SEQ ID NO:4; (2) a polypeptide comprising an aminoacid sequence having an identity of 90% or more to the amino acidsequence depicted in SEQ ID NO:4, and possessing an activity to inhibitthe motility of a sarcoma cell expressing a polypeptide comprising theamino acid sequence depicted in SEQ ID NO:2 when expressed in thesarcoma cell; and (3) a polypeptide comprising an amino acid sequenceresulting from deletion, substitution, insertion or addition of one or aplurality of amino acids in the amino acid sequence depicted in SEQ IDNO:4, and possessing an activity to inhibit the motility of a sarcomacell expressing a polypeptide comprising the amino acid sequencedepicted in SEQ ID NO:2 when expressed in the sarcoma cell.
 43. A methodfor inhibiting the motility of a sarcoma cell in a mammal, comprisingadministering to the mammal an effective amount of any polypeptideselected from among (1) to (3) below or an expression vector capable ofexpressing the polypeptide: (1) a polypeptide comprising the amino acidsequence depicted in SEQ ID NO:4; (2) a polypeptide comprising an aminoacid sequence having an identity of 90% or more to the amino acidsequence depicted in SEQ ID NO:4, and possessing an activity to inhibitthe motility of a sarcoma cell expressing a polypeptide comprising theamino acid sequence depicted in SEQ ID NO:2 when expressed in thesarcoma cell; and (3) a polypeptide comprising an amino acid sequenceresulting from deletion, substitution, insertion or addition of one or aplurality of amino acids in the amino acid sequence depicted in SEQ IDNO:4, and possessing an activity to inhibit the motility of a sarcomacell expressing a polypeptide comprising the amino acid sequencedepicted in SEQ ID NO:2 when expressed in the sarcoma cell.